The invention relates to a fluorescence bio-sensing method for detecting glutathione. According to the invention, a manganese dioxide nanometer material is used as an oxidizing agent and a recognition element; in the absence of glutathione (GSH), an oxidized o-diaminobenzene product is generated by catalytically oxidizing the manganese dioxide nanometer material for fluorescence detection; in the presence of the GSH, the manganese dioxide nanometer material is degraded by the GSH, the oxidizing capacity of manganese dioxide to o-diaminobenzene is inhibited and the fluorescence signal is weakened. The GSH detection is realized in the manner of comparing fluorescence values. Compared with the prior art, the method provided by the invention has the advantages of simple and convenient operation, high sensitivity, simplicity, low cost and specific detection for GSH. The detection method provided by the invention can be used for effectively detecting the GSH in a biological liquid sample.