Single-atom　catalysts　have　attracted　numerous　attention　due　to　the　high　utilization　of　metallic　atoms,　abundant　active　sites,　and　highly　catalytic　activities.　Herein,　a　single-atom　ruthenium　biomimetic　enzyme　(Ru-Ala-C3N4)　is　prepared　by　dispersing　Ru　atoms　on　a　carbon　nitride　support　for　the　simultaneous　electrochemical　detection　of　dopamine　(DA)　and　uric　acid　(UA),　which　are　coexisting　important　biological　molecules　involving　in　many　physiological　and　pathological　aspects.　The　morphology　and　elemental　states　of　the　single-atom　Ru　catalyst　are　studied　by　transmission　electron　microscopy,　energy　dispersive　X-ray　elemental　mapping,　high-angle　annular　dark　field-scanning　transmission　electron　microscopy,　and　high-resolution　X-ray　photoelectron　spectroscopy.　Results　show　that　Ru　atoms　atomically　disperse　throughout　the　C3N4　support　by　Ru-N　chemical　bonds.　The　electrochemical　characterizations　indicate　that　the　Ru-Ala-C3N4　biosensor　can　simultaneously　detect　the　oxidation　of　DA　and　UA　with　a　separation　of　peak　potential　of　180　mV　with　high　sensitivity　and　excellent　selectivity.　The　calibration　curves　for　DA　and　UA　range　from　0.06　to　490　and　0.5　to　2135　μM　with　detection　limits　of　20　and　170　nM,　respectively.　Moreover,　the　biosensor　was　applied　to　detect　DA　and　UA　in　real　biological　serum　samples　using　the　standard　addition　method　with　satisfactory　results.　©　2021　American　Chemical　Society.