A novel ultrasensitive biosensor was constructed by the [TBA]2[Ni(mnt)2]/graphene (mnt=maleonitriledithiolate) composite film for the detections of glutamate and alanine aminotransferase (ALT). The [TBA]2[Ni(mnt)2] nanocubes with a dimension of 500nm were in-situ crystallized on the surface of Au electrode modified using thiol graphene in advance. Due to the synergistic effect between the electrocatalysis of [Ni(mnt)2]2− anion and the excellent conductivity of graphene, the as-prepared biosensors showed remarkably reversible electrochemical redox and stability. It exhibited high sensitivities of 27.32μAmM−1cm−2 and 0.636nAμM−1 for the biosensing of glutamate and ALT with the corresponding wide linear ranges of 10–600μM−1 and 5–140UL−1, respectively. This high performance ALT biosensor is promising application in the trace analysis of more physiological substances, and the successful adoption of [TBA]2[Ni(mnt)2] material in the biosensor construction can arouse more dithiolene metal complexes in the further application of electrocatalysis.