H2S is the third endogenously generated gaseous signaling compound and has also been known to involve a variety of physiological processes. To better understand its physiological and pathological functions, efficient methods for monitoring of H2S are desired. Azide fluorogenic probes are popular because they can take place bioorthogonal reactions. In this work, by employing a fluorescein derivative as the fluorophore and an azide group as the recognition unit, we reported a new probe 5-azidofluorescein for H2S with improved sensitivity and selectivety. The probe shows very low background fluorescence in the absence of H2S. In the presence of H2S, however, a significant enhancement for excited fluorescence were observed, resulting in a high sensitivity to H2S in buffered (10 mmol/L HEPES, pH 7.0) aqueous acetonitrile solution (H2O/CH3CN = 1:3, v/v) with a detection limit of 0.035 μmol/L observed, much lower than the previously reported probes. All these features are favorable for direct monitoring of H2S with satisfactory sensitivity, demonstrating its value of practical application.