As a water-soluble vitamin, Vitamin B2 (VB2) is crucial for the health of living organisms. Therefore, developing sensitive and selective methods for detecting VB2 is essential for the quality control of food and pharmaceuticals as well as for clinical diagnosis. In this study, a cell-embedded living graphene hydrogel needle was prepared under ambient atmospheric conditions, where the electroactive bacteria Shewanella oneidensis MR-1 was used to induce the reduction of graphene oxide (GO) to graphene hydrogel under the confinement effect with a glass capillary tube. By using this living graphene hydrogel needle, an electrochemical biosensor for the detection of VB2 in microdroplet samples was developed. By taking advantage of a microscale graphene needle and electroactive S. oneidensis MR-1, this biosensor exhibited high sensitivity (LOD = 8.42 nM), excellent selectivity, and good anti-interference ability for amperometric detection of VB2 in a microdroplet (1 μL, the record-low sample volume). This work provided a reliable tool for trace detection of VB2 with minimized sample requirement, offering a sensitive and practical approach for food safety inspection and disease diagnosis with precious or limited samples.