• Enzyme-like Fe-N 5 single atom catalyst was employed for efficient sensing application. • Highly sensitive simultaneous sensing of dopamine and uric acid was realized. • The corresponding biosensor has been successfully applied for real human serum sample detection. Simultaneous highly sensitive and accurate detection of dopamine (DA) and uric acid (UA) is of great importance since they are important biomolecules that are commonly coexisted in the human body and play a crucial role in numerous physiological and pathological features. In this work, Fe-N 5 single atom catalyst (SAC) supported on graphene was firstly employed for simultaneous electrochemical detection DA and UA. Owing to its unique electronic structure and abundant active sites, under optimal conditions, the enzyme-like Fe-N 5 SAC constructed electrochemical (bio)sensor exhibits excellent linear characteristic responses in the range of 0.005–500 and 0.01–480 µM, as well as the low limit of detection of 0.007 and 0.027 nM for DA and UA, respectively. More critically, the developed electrochemical system shows promising merits of exceptional stability, good repeatability, and, consequently, it was successfully applied for simultaneous sensitive detection of DA and UA in human serum samples.