Stabilized aqueous solutions of α-chymotrypsin have a therapeutic utility in the treatment of certain forms of asthma, bronchitis, rhinitis, sinusitis, as well as certain dermatological conditions such as leg ulcers and ringworm. The aim of the present study was to investigate how lactose could influence the structure, thermal stability and the activity of α-chymotrypsin. The influence of lactose on the structure and stability of α-chymotrypsin (α-Chy) was explored using thermal stability, fluorescence spectroscopy, circular dichroism (CD) kinetic studies and molecular docking. We have calculated the thermodynamic parameters for the transition temperature (T_m), enthalpy change (ΔH°), entropy change (ΔS°) and Gibbs free energy change (ΔG°) to understand the stability of α-Chy. Lactose acted as an enhancer for the α-Chy stability, with varying efficacies and efficiencies. The results of the kinetic study displayed that the activity of α-Chy increased in the presence of lactose. The result reveals the ability of lactose to protect the native structural conformation of α-Chy. These results explicitly explain that stabilizing lactose is preferentially excluded from the surface of α-Chy, because water has a higher tendency toward favorable interactions with functional groups of α-Chy than with lactose. Fluorescence intensity changes showed static quenching during the lactose binding. The α-Chy fluorescence quenching suggested the more polar location of Trp residues. Near-UV and far-UV CD studies also proved the transfer of Trp, Phe and Tyr residues to a more flexible environment. Increasing of the α-Chy absorption in the presence of lactose was as a result of the formation of lactose–α-Chy complex. Molecular docking results revealed a negative value for the Gibbs free energy of the binding of lactose to α-Chy and the presence of one binding site. Docking study also revealed that hydrogen bond interactions dominated within the binding site.