In nanomedicine, efficiency of nano-medical drugs requires basic study of interactions between nanoparticles and red blood cell (RBC). The mechanical properties of RBC are important for blood microcirculation, which may get affected by nanoparticles. The main focus of the study is to probe gold nanoparticles (AuNP) interaction with single RBC using optical tweezer technique. AuNPs were selected due to their wide application in nanomedicine. In the present work, RBCs were suspended in the controlled volume of AuNP solution at different nanoparticle concentrations and incubated at 37 °C for different time periods. After the incubation, mechanical properties of RBC were investigated using single-beam optical tweezer. Additional information about RBC-AuNP interaction was obtained by UV-visible spectroscopy and Raman spectroscopy. Optical tweezer measurements results demonstrated that force required to trap RBC increases with increase in concentration of AuNP. Up to 40 nM concentration of AuNP, there were no significant effects on Soret band (409–413 nm), oxygenation state, and deformability properties of RBC. Folding time (the time required to trap the RBC completely when the trapping laser switches on) and unfolding time (the time required to recover the initial shape of RBC after the trapping laser switches off) of RBC did not change in the presence of AuNP. The UV-visible and Raman spectroscopy data are complementing with the optical trapping results. Gold nanoparticles do not affect the physiology of RBC up to 40 nM concentration of AuNP. Optical tweezer provides real-time insight which is a useful tool in nanomedicine, nanorobotic, and biomedical applications.