In this study, we report a new ratiometric electrochemical sensor to accurately and selectively detect hydrogen sulfide (H2S) in biological samples. In this system, it contains electroactive Ag dendritic nanocomposites (NCs) as a special recognition element of H2S and methylene blue (MB) as an inner reference molecular for a built-in correction to improve the accuracy. The Ag NCs are prepared and synthesized by a electrodeposition method, and the branches consist of rounded and smooth segments with a diameter of about 300 nm. In the presence of H2S, combining with the good adsorption ability of GO and more active sites of dendritic Ag nanostructure, the Ag NCs of GO/MB/Ag can be oxidized to Ag2S in the oxygen environment, resulting in the decrease of electrochemical oxidation peaks of Ag (0.42 V) and constant of current responses of MB. On the basis of the ratio of IAg/IMB at two distinct potentials, the developed ratiometric electrochemical sensor displays a wide linear range from 30 to 500 μM with high accuracy, selectivity, and stability. This strategy provides a reliable and feasible method for the detections of H2S in real samples, including biological samples related to physiological and pathological events of hydrogen sulfide production.