A hydroxyphenylbenzothiazole (HBT)-based fluorescent probe (HBT-Py) was synthesized to selectively and simultaneously detect GSH and Arg in NIR (684 nm) and blue (469 nm) regions excited by 400 nm at the absolutely same measured conditions, respectively. After studied the detected mechanism through TLC, HRMS and optical data, we proposed that the detection for GSH could result from the Michael addition and esterlysis reactions between GSH and the probe, followed by ESIPT process to get the fluorescent signal at 684 nm. While the mechanism for detection Arg could be from esterlysis and ESIPT-block due to the strong basicity and formation of hydrogen bond from guanidine group in Arg.