Alanine (Ala), as the most important free amino acid, plays a significant role in food taste characteristics and human health regulation. The feasibility of using near–infrared hyperspectral imaging (NIR–HSI) combined with two–dimensional correlation spectroscopy (2D–COS) analysis to predict beef Ala content quickly and nondestructively is first proposed in this study. With Ala content as the external disturbance condition, the sequence of chemical bond changes caused by synchronous and asynchronous correlation spectrum changes in 2D–COS was analyzed, and local sensitive variables closely related to Ala content were obtained. On this basis, the simplified linear, nonlinear, and artificial neural network models developed by the weighted coefficient based on the feature wavelength extraction method were compared. The results show that with the change in Ala content in beef, the double-frequency absorption of the C-H bond of CH2 in the chemical bond sequence occurred prior to the third vibration of the C=O bond and the first stretching of O-H in COOH. Furthermore, the wavelength within the 1136–1478 nm spectrum range was obtained as the local study area of Ala content. The linear partial least squares regression (PLSR) model based on effective wavelengths was selected by competitive adaptive reweighted sampling (CARS) from 2D–COS analysis, and provided excellent results (R2C of 0.8141, R2P of 0.8458, and RPDp of 2.54). Finally, the visual distribution of Ala content in beef was produced by the optimal simplified combination model. The results show that 2D–COS combined with NIR–HSI could be used as an effective method to monitor Ala content in beef.