Analysis of leaf reflectance can provide a rich source of information about plant physiological process, surface properties of the leaf, as well as internal structure. In the present paper, the changes of reflectance spectra and spectral indices for transgenic barley and non-transgenic control variety based on measurements of single leaf were studied. The results showed that the ratio spectra that calculated by dividing one reflectance spectrum of transgenic barley by its’ non-transgenic counterpart spectra were better to illustrate fine differences among spectra of leaves from different barley varieties. Ratio spectra varied between each pairs, but for most ratio spectrum indicated the reflectance differences between transgenic barley and non-transgenic counterpart were most obvious in the red spectrum around the chlorophyll peak and red edge (650~700 nm) and nearly identical in the green spectrum near 550 nm and within the near infra-red region (beyond 750 nm). Across the entire calculated spectrum, the changes of reflectance within the region from 650 to 700 nm seem to be very sensitive to respond to possible unintended effects of genetic modification. Six published reflectance indices, the red edge (λRE), the modified normalized difference vegetation index (mND), the structure-independent pigment index (SIPI), the ratio of reflectance in the red and green regions of the spectrum (RRed/RGreen), the photochemical reflectance index (PRI) and the near-infrared reflectance at 800 nm (NIRR800) were used to make comparisons possible between species. The analyses of changes of these indices indicated that they were highly dependent on growth stages and barley varieties. Once indices described above have significant changes will mean that photosynthetic process, function and status of plants may be changed.