Dopamine (DA) and uric acid (UA) are essential for many physiological processes in the human body. Abnormal levels of DA and UA can lead to multiple diseases, such as Parkinson’s disease and gout. In this work, a three-dimensional reduced graphene oxide–MXene (3D rGO-Ti₃C₂) composite electrode was prepared using a simple one-step hydrothermal reduction process, which could separate the oxidation potentials of DA and UA, enabling the simultaneous detection of DA and UA. The 3D rGO-Ti₃C₂ electrode exhibited excellent electrocatalytic activity towards both DA and UA. In 0.01 M PBS solution, the linear range of DA was 0.5–500 µM with a sensitivity of 0.74 µA·µM⁻¹·cm⁻² and a detection limit of 0.056 µM (S/N = 3), while the linear range of UA was 0.5–60 µM and 80–450 µM, with sensitivity of 2.96 and 0.81 µA·µM⁻¹·cm⁻², respectively, and a detection limit of 0.086 µM (S/N = 3). In 10% fetal bovine serum (FBS) solution, the linear range of DA was 0.5–500 µM with a sensitivity of 0.41 µA·µM⁻¹·cm⁻² and a detection limit of 0.091 µM (S/N = 3). The linear range of UA was 2–500 µM with a sensitivity of 0.11 µA·µM⁻¹·cm⁻² and a detection limit of 0.6 µM (S/N = 3). The modified electrode exhibited advantages such as high sensitivity, a strong anti-interference capability, and good repeatability. Furthermore, the modified electrode was successfully used for DA measurement in vivo. This could present a simple reliable route for neurotransmitter detection in neuroscience.