Chlorophyll content of cucumber leaf in vivo was detected by reflectance laser-induced fluorescence (LIF) spectrum analysis technique in this research. Fluorescence spectrums, which was induced by four power (2.5 mW，5.0 mW，7.5 mW，10.0 mW) of center wavelength 473 nm and 660 nm laser respectively, were analyzed in the experiment. Results showed that the fluorescence spectrum has a favorable precision and stability under the condition of 7.5 mW and 473 nm laser, and there is a significant linear correlation between the ratio of chlorophyll fluorescence at 732 nm to 685 nm (F732/F685) and the chlorophyll content. Furthermore the regression model was established (R2>0.93, p<0.001) in this excitation light source condition. This research doesn’t only extend the scope of the application of chlorophyll fluorescence detection technology, but also provides a theoretical basis of the development of portable and non-destructive plants chlorophyll content detection equipment. 