||pH-responsive fluorescent carbon nanoparticles (FNPs) combined with fluorescent, colorimetric, and electrochemical signals generated from coated surfaces, we have discovered a fast and convenient method to observe antifouling performance of FNP-coated surfaces with mammalian cell and bacterial based on fluorescent off/on signal. FNPs were carbonized from zwitterionic polymer, which is coatable on various surfaces and highly sensitive in a broad range of pH. The coated surfaces showed excellent antifouling effect of mammalian cells adhesion and exhibited a strong interaction with bacteria surface as observed in a fluorescence behavior. FNP-modified electrode showed a decrease in electrochemical current attributed high sensitivity to biofouling formation in various pH. In conclusion, a pH-switchable zwitterionic FNP-coated surface is introduced as a fast and accurate method for detection and control the biological adhesion onto diverse surfaces based on fluorescent behavior.