In this study, a series of variously substituted 2,3-dihydroquinolin-4-imines (DQIs) were synthesized from N-substituted propargylanilines by copper(I)-catalyzed annulation in good isolated yields of 63–88%. The new and mild synthetic strategy allows a rapid collection of various substituted DQIs. Surprisingly, DQIs thus obtained exhibited bright fluorescence with quantum yields up to 66% along with substituent-dependent emission color. An unusual solvatochromic effect is observed, considerably diminishing the quantum yield from 62% in dichloromethane to 6% in methanol, as well as a significant red-shift of emission from 478 nm in n-hexane to 573 nm in methanol. The density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations were performed for understanding the excited-state nature of DQI system. Accordingly, a tailored DQI 2aa was designed and synthesized to give emission at 559 nm with an obvious red-shift of 59 nm compared to the 7-methoxy substituted DQI. The unique environment-sensitive property makes DQI an ideal fluorescent turn-on biosensor for selective biomolecular recognition. An aryl sulfonamide conjugated DQI (SA-DQI) molecule has successfully used in the detection of carbonic anhydraes II (CAII) with a fifteen times enhancement of fluorescent intensity. Remarkably, the SA-DQI is a background-free fluorescent probe in the recognition of membrane bound CAII on 293T cells without removing unbound SA-DQIs. In this research, we first report a new environment-sensitive DQI fluorophore family with customized photophysical property. The easy structural modification and background-free biomolecular labeling of DQI can certainly be a powerful tool in the real-time study of biomolecular interactions.