In this study, a two-section clinical sample detection on viscosity and bimolecule sensing technique based on measuring the Brownian velocity of nanobeads has been successfully developed. Bare and antibody-coated fluorescent nanobeads were used to detect various viscous solutions and different concentrations of bio-molecular samples, respectively. The Brownian velocities of the nanobeads became slower when the sizes of antibody-coated nanobeads increased during the antibody-antigen interaction process or when the bare nanobeads were seeded in a higher viscosity solution. In order to obtain the characteristics of the Brownian motion of nanobeads in the static liquid, total internal reflection fluorescent velocimetry (TIRFV) was employed. This sensing technique should be useful in clinical diagnosis, bio-molecule judgment, and the study of fluid characteristics.