Pitaya is a new kind of fruit with high nutritional value and good economic benefit which was introduced into China for a short time. Its stems are the most important photosynthetic organs, which is quite different from the common green leaf fruit trees. In order to explore the spectral characteristics and the estimation method of biochemical components of vegetation using stems for photosynthesis, the field experiments were carried out at four nitrogen application levels in Luodian Guizhou, the chlorophyll content of Hylocereus polyrhizus stems were taken as the research object. Firstly, hyperspectral reflection data and chlorophyll content data of Hylocereus polyrhizus stems under different nitrogen nutrient were measured simultaneously; Secondly, the hyperspectral data were analyzed by mathematical transform, continuous wavelet transform(CWT)and correlation analysis algorithm to extract and screen the characteristic bands; Finally, the chlorophyll content estimation model of stem was established by partial least squares regression(PLSA). The results showed that: (1)The overall trend of the original spectral curve of Hylocereus polyrhizus stems is similar to common green leafed plants, the bands sensitive to chlorophyll content of branches are mostly located in the red edge and near-infrared region. In the near-infrared region, the variation of stems spectrum with nitrogen application is different from that of green leaves. The absorption peak (valley) of Hylocereus polyrhizus branches spectrum increased (deepened) with the increase of nitrogen application. (2)First derivative(FD)and CWT in the scale of L₁—L₅ can effectively improve the sensitivity of the spectrum to chlorophyll content. The sensitive region of the original spectrum and chlorophyll content of Hylocereus polyrhizus stems is located in 730~1 400 nm. Both the mathematical transform and CWTcan significantly improve the sensitivity of the spectrum to chlorophyll content, but the distribution of sensitive bands is relatively scattered, and there are more sensitive bands in the red edge (730 nm) and near infrared region(1 100~1 600 nm), which is different from the distribution of chlorophyll content sensitive bands in leaves. (3)Both the mathematical transformation and CWT can significantly improve the spectral estimation ability of chlorophyll contentin Hylocereus polyrhizus stems. The estimation model based on FD the optimal models of mathematical transformation, the verification accuracy is $R_{verification}^{2}$=0.625, RMSE=0.048, RPD=1.238(FD). The model based on L1 and L4 has relatively high modeling accuracy and estimation accuracy, which is the best model with $R_{verification}^{2}$=0.678, RMSE=0.037, RPD=1.652(CWT). Hyperspectral technology can be used as a non-destructive monitoring method for chlorophyll content and nutrition diagnosis of Pitaya. This study provides a supplement for improving the retrieval of chlorophyll content of different vegetation types based on hyperspectral index.