The interactions between gemcitabine hydrochloride (GEM) and bovine serum albumin (BSA) or human serum albumin (HSA) have been studied by spectroscopic techniques. By the analysis of fluorescence spectrum and fluorescence intensity, it was observed that the GEM has a strong ability to quench the intrinsic fluorescence of both BSA and HSA through a static quenching procedure. The association constants of GEM with BSA and HSA were determined at different temperatures based on fluorescence quenching results. The negative ΔH° and positive ΔS° values in case of GEM–BSA and GEM–HSA complexes showed that both hydrogen bonds and hydrophobic interactions play a role in the binding of GEM to BSA or HSA. Experimental results showed that the binding of GEM to BSA or HSA induced conformational changes in BSA and HSA. From the quantitative analysis data of CD spectra, the α-helix of 57.58% and 34.82% in free BSA and free HSA decreased to 40.82% and 29.84% in BSA–GEM and HSA–GEM complexes, respectively, and hence confirmed that the secondary structure of protein was altered by GEM. The interactions of BSA and HSA with GEM were also confirmed by UV absorption spectra. The distance, r, between donor (BSA or HSA) and acceptor (GEM) was obtained according to the Förster's theory of non-radiation energy transfer. The effects of common ions on the binding constants of both BSA–GEM and HSA–GEM complexes were also investigated.