Accurate detection of rice nitrogen content is an important aspect of precision fertilization in rice fields. Nitrogen content variation of rice leaves will cause changes in emissivity of leaves and canopy. Hyperspectral remote sensing is one of the key technologies for non-destructive monitoring of crop nitrogen. This study focuses on the study of nitrogen content monitoring and the sensitive band’s selection through machine learning methods based on 2-year rice nitrogen fertilization experiments carried out in Jianli Hubei during 2018—2019. Hyperspectral reflectance spectral data at the leaf and canopy level and the corresponding leaf nitrogen content data were collected at rice tillering, jointing, booting, flowering and filling stage, respectively. Correlation analysis and Gaussian process regression (GPR) were used to select nitrogen sensitive bands for raw spectra and first-order derivative reflectance (FDR) spectra in rice leaves and canopy level. The nitrogen content estimation models were then constructed through single-band regression analysis, Random Forest (RF) and Support Vector Regression (SVR) method for rice raw spectra data. The Gaussian Process Regression-Random Forest (GPR-RF), Gaussian Process Regression- Support Vector Regression (GPR-SVR), and GPR method were also used to construct the nitrogen estimation model for the nitrogen-sensitive selection bands. The results showed that the GPR method’s sensitive bands were consistent with variation rule of the nitrogen content and spectral changes in rice. The leaf-level model’s over all accuracy was higher than that of the canopy-level model under the same conditions while using FDR spectra was more accurate at canopy level for it could attenuate the effect of background noise in the rice field. R2 of calibration datasets and validation sets are 0.79 and 0.84 at the leaf level, while 0.80 and 0.77 at canopy level. Compared with the correlation regression model, the machine learning methods were less affected by rice growth stages (R2>0.80, NRMSE<10%). RF was more suitable than SVR for modeling GPR-selection nitrogen sensitive bands, and the GPR-RF model can use about 1.5% of the bands to reach the accuracy of the RF model using all the bands. The GPR model works well on nitrogen estimation through nitrogen -sensitive bands at leaf and canopy level, not only for the full-growth stage but also for the single-growth stage(R2>0.94, NRMSE<6%). Besides, compared with the other four machine learning methods, the GPR model can improve the accuracy and stability of the estimation of nitrogen content at the canopy level that R2 increased by 0.02 and NRMSE decreased by 1.2%. GPR method provides a methodological reference for selecting crop nitrogen hyper-spectrally sensitive bands and inversion of the nitrogen content of leaves and canopy level during rice different growth period.