ABSTRACT Herein, we report a new type of non-enzymatic nanostructured biosensor designed for the concurrent determination of dopamine, cholesterol, and glucose in human blood samples. The prepared biosensor using low-temperature one-pot hydrothermal processing is a Zn-doped SnO2-dispersed nitrogen-doped reduced graphene oxide (N-rGO)/Zn-SnO2 nanocomposite. This modified electrode allows direct electron transfer and thus offers electrochemical techniques to detect target molecules in analytes without the use of enzymes. The biosensor displayed good voltammetric responses within the human reference range: 20–70 nM for dopamine, 1–8 mM for cholesterol, and 1–10 mM for glucose. It also showed excellent reusability and can be restored by washing with ethanol. Due to the use of Zn-doped SnO2 nanoparticles for enhanced catalytic activity and N-rGO for high conductivity and active sites, sensitive and selective detection can be achieved. This hybrid composite demonstrates excellent catalytic activity, repeatability, stability, and clinical comparability for real-time measurement of dopamine, cholesterol, and glucose simultaneously.