Abstract: α7 nicotinic acetylcholine receptor (nAChR) is widely distributed in the central and peripheral nervous systems, and is closely related to a variety of neurological diseases and inflammation response. α-ConotoxinA10LPnIA, as an antagonist targeting α7 nAChR, plays an important role in studying the physiological and pathological processes involved in α7 nAChR.A10LPnIA was labeled with fluorescein 5-carboxytetramethylrho-damine, and the active peptide (A10LPnIA-F) was obtained by a two-step oxidative folding procedure in vitro. The Xenopus oocyte expression system and the two-electrode voltage clamp technique were used to identify the potency ofA10LPnIA-F fluorescent peptide, and its cytotoxicity was detected by mouse macrophages and CCK8 method. The molecular weight ofA10LPnIA-F fluorescent peptide was identified by mass spectrometry as 2 077.28 Da, which was consistent with the theoretical value. Electrophysiological determination of its halfmaximal inhibitory concentration (IC₅₀) for α7 nAChR is 17.32 nmol·L^-1, which is consistent withA10LPnIA (IC₅₀, 13.84 nmol·L^-1). The cytotoxicity test results showed that within the concentration range of 5 nmol·L^-1 to 10 μmol·L^-1, there was no significant inhibition on the growth of mouse macrophages. The results showed that the α-conotoxin fluorescent probeA10LPnIA could provide pharmacological tools for the research of α7 nAChRrelated neurophysiological and pathological mechanisms.