This paper sheds light into numerous features of the interaction of copper sulfide nanoparticles (CuS NPs) with vital proteins viz., bovine serum albumin (BSA), human serum albumin (HSA) and collagen in the absence and presence of different surfactants. We used the wet chemical co-precipitation process for the synthesis of CuS NPs. The synthesized NPs were characterized through fourier transform infrared Spectroscopy (FTIR), UV–visible spectroscopy, X-ray diffraction (XRD), zeta potential, dynamic light scattering (DLS), scanning electron microscopy (SEM), elemental mapping and energy dispersive X-ray spectroscopy (EDAX). CuS NPs-protein interactions were analyzed by fluorescence spectroscopy, circular dichroism spectroscopy (CD), UV–visible spectroscopy, DLS technique, sodium dodecyl sulphate -polyacrylamide gel electrophoresis (SDS -PAGE). The esterase activity and cysteine reactivity of BSA was checked in association with CuS NPs and surfactants. Furthermore, the radical scavenging activity of synthesized NPs was also tested (DPPH assay). Interaction of CuS NPs with DNA alone and in presence of surfactants was studied by gel electrophoresis. CTAB, DTAB and Tween 80 enhanced CuS NPs-protein binding. Tween 80 was furthermore found to impart constant biocompatibility to CuS NPs. The esterase and cysteine activity of BSA was found to remain unaffected in presence of Tween 80 only. Moreover, the antioxidant activity studies by DPPH assay revealed maximum radical scavenging activity for Tween 80 based systems. Fluorescence studies at 298, 303, 313, 319 and 327 K showed enhanced strength and association between CuS NPs and BSA in presence of Tween 80. The obtained thermodynamic parameters for NPs-protein bindings at various temperatures suggest the dominance of hydrogen bonding and Vander Waals forces between CuS NPs/Tween 80 and BSA systems. The present study evaluates the effects of surfactant coated CuS NPs on the vital biomolecules. The study thus possess potential in designing stable and biocompatible CuS NPs for biomedical utilizations.