Human serum albumin (HSA) accounts for 60% of total plasma protein and is closely related to life-threatening diseases. In this strategy, three fluorescent sensors of Nap-OH, Nap-C and Nap-NO2 based on twisted intramolecular charge transfer (TICT) are designed and synthesized for detecting of HSA, and the effect of electron donating-withdrawing groups of sensors on the detection of HSA is studied. After added HSA, the fluorescent emission of Nap-NO2 is turned on at 630 nm, and the color changed from colorless to pink. Nap-NO2 is characterized by a 14.06-fold increase in fluorescence emission and has high sensitivity, good selectivity, fast response, and good stability. Nap-NO2 could effectively distinguish HSA from other proteins, including bovine serum albumin (BSA). Drug competition experiments are performed that Nap-NO2 can bind to the IB domain of HSA, avoiding the interference of commonly used drugs. Nap-OH, Nap-C and Nap-NO2 can also be used for viscosity detection.