Fluorescence correlation spectroscopy (FCS) has been adopted to investigate the conformational transition of poly(N-isopropylacrylamide) (PNIPAM) single chains with moderate molecular weights in the cononsolvency. process. A practical approach of performing accurate FCS measurements with the presence of the refractive index mismatch was developed. The practical and reliable FCS calibration facilitates the acquisition of the hydrodynamic radius (R-H) of PNIPAM single chains with the change of the water-ethanol composition. By using the synthesized PNIPAM samples covering a range of degrees of polymerization (N), the scaling analysis in the relationship of R-H similar to N-nu exhibits a progressive, re-entrant change of the scaling index (nu) between good solvent (0.57) and poor solvent (similar to 1/3) condition, which is a reflection of a re-entrant conformational transition of the polymers. Furthermore, the highly asymmetrical feature of the cononsolvency process of single PNIPAM chains was unveiled, which indicates a much stronger effect or interaction of the ethanol molecules to the PNIPAM chain. Comparisons of the present results with previous reports provided new information to the mechanism model of the PNIPAM cononsolvency.