A measurement method based on the floating reference position is proposed, and the in vivo relative measurement of glucose concentration can be expected. The function of floating reference position is analyzed based on the analytical solution to the steady state diffuse equation in a semi-infinite medium. Moreover, the distribution of floating reference position for the three-layer skin model within the wavelength of 1000-1400 nm is investigated based on the Monte Carlo simulation, as well as the influence of absorption coefficient, scattering coefficient, and thickness of epidermis and dermis on floating reference position. A customized optical fiber system with multiple circles based on the superluminescent diode is presented for initial validation of the existence of floating reference position in human body. The simulation results show that the reference positions of the three-layer skin model exist in the wavelength range of 1000-1400 nm and are wavelength dependent, the influence of scattering coefficient is stronger than that of absorption coefficient, and the influence of epidermis thickness variation is stronger than that of dermis thickness variation. The in vivo experiments on three volunteers show that floating reference positions of human palm at 1050, 1219, 1314 nm exist, and are located within the radial distance from 1.25 mm to 1.75 mm. The experimental results are lower than the simulation results, with a maximum bias of 0.75 mm. The result is valuable to the design of optical probe applied in the floating reference measurement.