HSA (human serum albumin), a most abundant protein in blood serum, plays a key role in maintaining human health. Abnormal HSA level is correlated with many diseases, and thus has been used as an essential biomarker for therapeutic monitoring and biomedical diagnosis. Development of small-molecule fluorescent probes allowing the selective and sensitive recognition of HSA in in vitro and in vivo is of fundamental importance in basic biological research as well as medical diagnosis. Herein, we reported a series of new synthesized fluorescent dyes containing D-π-A constitution, which exhibited different optical properties in solution and solid state. Among them, dye M-H-SO with a hydrophilic sulfonate group at electron-acceptor part displayed selectivity for discrimination of HSA from BSA and other enzymes. Upon binding of dye M-H-SO with HSA, a significant fluorescence enhancement with a turn-on ratio about 96-fold was triggered. The detection limit was estimated to be ∼ 40 nM. Studies on the interaction mechanism revealed that dye M-H-SO could bind to site III of HSA with a 1:1 binding stoichiometry. Furthermore, dye M-H-SO has been applied to determine HSA in real urine samples with good recoveries, which provided a useful method for HSA analysis in biological fluids.