In order to improve accuracy of quantitative analysis model for the greenhouse tomato nitrogen, phosphorus and potassium nutrient stress, and explore the advantages of polarization non-destructive detection in single-leaf plants scale, polarized reflectance characteristics of greenhouse nutrient deficiency tomato leaves in different growing seasons and different deficiency extents were both examined via means of polarized reflectance spectroscopy system, which was self-developed by the research group. The main factors with effects on the polarized reflectance characteristics of tomato leaves were discussed, such as incident zenith angle, azimuth angle, detection zenith angle, light source polarizer degree, and detector polarizer degree. Experiments were carried out to verify the optimum level of above five parameters by means of range analysis of orthogonal experiments, through that way we can know the best angle combination of five parameters. Based on the above analysis, the angle combination and sorting of detecting tomato nutrients deficiency leaves via means of polarization spectroscopy system were obtained as follows: incident zenith angle 60°, light source polarizer degree 0°, detection zenith angle 45°, detector polarizer degree 45° and azimuth angle 180°. At the same time, both the spectra of nitrogen, phosphorus and potassium deficiency leaves in different growth stages and different deficiency extent leaves were compared with each other. Results show that there is a positive correlation between the greenhouse nutrient deficiency tomato leaves growth cycle and tomato leaves polarized reflectance spectra. Nutrient excess or nutrient deficiency can both lead to polarized reflectance decline and polarized reflectance decline extent of greenhouse tomato leaves is more obvious during the fruiting and harvest period. This paper has a certain theoretical and practical significance in the research on nutrition rapid detection on the plant single leaf scale by means of polarized reflectance spectrum.