Leaf chlorophyll content (LCC) is crucial for monitoring the physiological processes of crops. Many studies have utilized spectral features to develop regression models for accurate LCC estimation, enabling the quantitative assessment and evaluation of crop growth status. The selection of optimal spectral features and regression algorithms significantly affects the precision of LCC estimation. In this study, we compared and analyzed the optimal spectral features for LCC estimation, as well as the consistency of machine learning methods across different crop types, phenology periods, and sensors. First, we extracted various spectral features, including the original spectral features (OS), first-order derivative spectral features (FDS), original continuum-removed spectra (CR) along with their four related derivative spectral features, principal component variables derived from different spectral features, and highly correlated spectral features with LCC. These extracted spectral features were then employed to construct the LCC models using six common regression algorithms on different datasets. Finally, we analyzed the optimal combination of spectral features and regression algorithms for accurate LCC estimation considering various dimensions, such as crop type, phenological period, and sensor used in observation conditions. The results demonstrate that the combinations of the principal component variables of continuum-removed derivative reflectance with the top 10 correlations with LCC (PCA_CRDR_R) combined with Gaussian process regression (GPR) can be considered as the optimal choice for estimating LCC under diverse observation conditions at a canopy scale, and its R2 can reach 0.62 for sugar beet LCC estimation; thus providing valuable theoretical guidance for selecting appropriate spectral features for LCC estimation.