The influence of the equidistant sampling method was explored in a hyperspectral model for the accurate prediction of the water content of apple tree canopy. The relationship between spectral reflectance and water content was explored using the sample partition methods of equidistant sampling and random sampling, and a stepwise regression model of the apple canopy water content was established. The results showed that the random sampling model was Y = 0.4797 - 721787.3883 × Z3 - 766567.1103 × Z5 - 771392.9030 × Z6; the equidistant sampling model was Y = 0.4613 - 480610.4213 × Z2 - 552189.0450 × Z5 - 1006181.8358 × Z6. After verification, the equidistant sampling method was verified to offer a superior prediction ability. The calibration set coefficient of determination of 0.6599 and validation set coefficient of determination of 0.8221 were higher than that of the random sampling model by 9.20% and 10.90%, respectively. The root mean square error (RMSE) of 0.0365 and relative error (RE) of 0.0626 were lower than that of the random sampling model by 17.23% and 17.09%, respectively. Dividing the calibration set and validation set by the equidistant sampling method can improve the prediction accuracy of the hyperspectral model of apple canopy water content.