In this study, we developed a portable smartphone-based diffusometry for analyzing the C-reactive protein (CRP) concentration. An optimized fluorescence microscopic add-on system for a smartphone was used to image the 300 nm fluorescent beads. Sequential nanobead images were recorded for a period and the image data were used for fluorescence correlation spectrometric (FCS) analysis. Through the analysis, the nanobeads' diffusion coefficient was obtained. Further, the diffusion coefficients of the anti-CRP-coated nanobeads, which were suspended in the samples with various CRP concentrations, were estimated using smartphone-based diffusometry. After 10 min of reaction, the anti-CRP-coated nanobeads in a higher CRP concentration solution led to a lower diffusion coefficient. Based on the experiments, a linear sensing range of 1~8 µg/mL was found.