It is highly necessary to apply unmanned aerial vehicle (UAV) remote sensing technology to forest health assessment. To prove the feasibility of quantitative inversion of photosynthetic pigment content (PPC) in Populus euphratica Oliv. individual tree canopy (PeITC) by using multispectral UAV images, in this study, Parrot Sequoia+ multispectral UAV system was manipulated to collect the images of Populus euphratica (Populus euphratica Oliv.) sample plots in Daliyabuyi Oasis from 2019 to 2020, and the canopy PPCs of five Populus euphratica sample trees per plot were determined in six plots. The Populus euphratica crown regions were extracted by grey wolf optimizer-OTSU (GWO-OTSU) multithreshold segmentation algorithm from the normalized difference vegetation index (NDVI) images of Populus euphratica sample plots obtained after preprocessing, and the PeITCs were segmented by multiresolution segmentation algorithm. The mean values of 27 spectral indices in the PeITCs were calculated in each plot, and the optimal model was constructed for quantitative estimation of the PPCs in the PeITCs, then the inversion results were compared and verified based on GF-6 and ZY1-02D satellite imageries respectively. The results were as follows. (1) The average value of canopy chlorophyll content (Chl) was 2.007 mg/g, the mean value of canopy carotenoid content (Car) was 0.703 mg/g. The coefficient of variation (C.V) of both were basically the same and they were both of strong variability. The measured PPCs of the PeITCs in Daliyabuyi Oasis was generally low. The average contents of chlorophyll and carotenoid in PeITC in June were more than twice those in August, while the mean ratio between them was significantly lower in June than in August. The measured PPCs had no obvious spatial distribution law. However, that could prove the rationality of sample selection in this study. (2) NDVI had the best effect of highlighting vegetation among all quadrats in the study area. Based on the GWO-OTSU multithreshold segmentation method, the canopy area of Populus euphratica could be quickly and effectively extracted from the quadrat NDVI map. The best segmentation effect of PeITCs was obtained based on a multiresolution segmentation method when the segmentation scale was 120, the shape index was 0.7, and the compactness index was 0.5. Compared with manual vectorization method of visual interpretation, the root mean square error (RMSE) and Pearson correlation coefficient (R) values of the mean NDVI values in PeITCs obtained by these two methods were 0.038 and 0.951. (3) Only 12 of the 27 spectral indices were significantly correlated with Chl and Car at the significance level of 0.02. Characteristics of the calibration set and validation set were basically consistent with those of the entire set. The classification and regression tree-decision tree (CART-DT) model performed best in the estimation of the PPCs in the PeITCs, in which, when estimating the Car, the calibration coefficient of determination (R2C) was 0.843, the calibration root mean square error (RMSEC) was 0.084, the calibration residual prediction deviation (RPDC) was 2.525, the validation coefficient of determination (R2V) was 0.670, the validation root mean square error (RMSEV) was 0.251, the validation residual prediction deviation (RPDV) was 1.741. (4) Qualitative comparison of spectral reflectance and NDVI values between GF-6 multispectral imagery and Parrot Sequoia+ multispectral image on the 172 PeITCs can show the reliability of Parrot Sequoia+ multispectral image. The comparison results of five PeITCs relative health degree judged by field vision judgment, measured SPAD value, predicted value of Chl (Chlpre), the red edge value calculated by ZY1-02D (ZY1-02Dred edge) and the Carotenoid Reflection Index 2 (CRI2) value calculated by ZY1-02D (ZY1-02DCRI2) can further prove the scientificity of inversion results to a certain extent. These results indicate that multispectral UAV images can be applied for quantitative inversion of PPC in PeITC, which could provide an indicator for the construction of a Populus euphratica individual tree health evaluation indicator system based on UAV remote sensing technology in the next step.