A new sensitive and selective two-photon fluorescent chemosensor L bearing triphenylamine skeleton was conveniently prepared for detecting homocysteine (Hcy) through Cu2+ displaced method in situ system generated from the pre-prepared L-Cu2+. Furthermore, Cu2+ of the polluted water can also be monitored conveniently in range from 10(-8) to 10(-2) M by naked eye with test strips coated with L. What's more, L having low cytotoxicity and good biocompatibility were successfully applied to imaging for endogenously and exogenously tracking Hcy in HeLa cell by two-photon fluorescence microscopy.