Cotton yield estimation is of great practical significance to producers, allowing them to make rational management decisions. At present, crop yield estimation methods mainly comprise traditional agricultural yield estimation methods, which have many shortcomings. As an ideal “probe” for detecting crop photosynthesis, sun-induced chlorophyll fluorescence (SIF) can directly reflect the dynamics of actual crop photosynthesis and has the potential to predict crop yield, in order to realize cotton yield estimation based on canopy SIF. In this study, we set up field trials with different nitrogen fertilizer gradients. The changes of canopy SIF and the physiological parameters of cotton in different growth periods were analyzed. To investigate the effects of LAI and AGB on canopy SIF estimation of cotton yield, four algorithms, Ada Boost (Adaptive Boosting), Bagging (Bootstrap Aggregating), RF (Random Forest), and BPNN (Backpropagation Neural Network), were used to construct cotton yield estimation models based on the SIF and SIFy (the normalization of SIF by incident photosynthetically active radiation) for different time and growth periods. The results include the following: (1) The effects of the leaf area index (LAI) and aboveground biomass (AGB) on cotton canopy SIF and cotton yield were similar. The correlation coefficients of LAI and AGB with cotton yield and SIF were significantly positively correlated with each other starting from the budding period, reaching the maximum at the flowering and boll period, and decreasing at the boll period; (2) In different monitoring time periods, the R2 of the cotton yield estimation model established based on SIF and SIFy showed a gradual increase from 10:00 to 14:00 and a gradual decrease from 15:00 to 19:00, while the optimal observation time was from 14:00 to 15:00. The R2 increased with the progression of growth from the budding period to the flowering and boll period and decreased at the boll period, while the optimum growth period was the flowering and boll period; (3) Compared to SIF, SIFy has a superior estimation of yield. The best yield estimation model based on the RF algorithm (R2 = 0.9612, RMSE = 66.27 kg·ha−1, RPD = 4.264) was found in the canopy SIFy of the flowering and boll period at 14:00–15:00, followed by the model utilizing the Bagging algorithm (R2 = 0.8898) and Ada Boost algorithm (R2 = 0.8796). In summary, SIFy eliminates the effect of PAR (photosynthetically active radiation) on SIF and can further improve the estimation of SIF production. This study provides empirical support for SIF estimation of cotton yield and methodological and modeling support for the accurate estimation of cotton yield.