The interaction between rolitetracycline (RTC) and human serum albumin (HSA) has been investigated by using multi-spectral methods and molecular modeling under physiological conditions. Fluorescence spectra results revealed the presence of static quenching mechanism in the binding of RTC to HSA. The binding constants were of K298 K=3.13×105 L·mol-1 and K310 K=0.70×105 L·mol-1, respectively. The numbers of binding site were 1.09 and 0.95 at two different temperatures. The thermodynamic parameters indicated that hydrogen bond and van der Waals force were the major driving forces for interaction between RTC and HSA. The binding distance of RTC to HSA was calculated to 2.59 nm based on fluorescence resonance energy transfer. UV-Vis absorption spectra and 3-D fluorescence demonstrated that the conformation and micro-environment of HSA were changed with the addition of RTC. FT-IR spectra was used to quantitatively calculate the alternations of secondary structure of HSA with the α-helices content reduction from 51.5% to 43.1%, increasing the content of β-sheet (30.3%～31.4%) and β-turn (15.6%～16.1%). According to molecular modeling studies and site marker competitive experiments, it indicated that RTC bound in the Sudlow’s drug binding site Ⅰ of HSA and the subdomain ⅡA where Lys195, Arg218 and Arg222 residues were located. The work is helpful to understand the interaction mechanism between RTC and HSA at molecular level.