A glassy carbon electrode was modified with a 3D-networked nanostructure composed of MoS, reduced graphene oxide and gold nanoparticles (3D-MoS/rGO/Au). The composites were prepared through in-situ growth of gold nanoparticles on 3D-MoS/rGO nanosheets via a hydrothermal method. The morphology and electrochemical features of the composite were investigated. The 3D-MoS/rGO/Au sensor exhibits excellent electrocatalytic activity for simultaneous detection of ascorbic acid (AA), dopamine (DA) and uric acid (UA). The oxidation potentials are well separated at around -0.05 V for AA, 0.06 V for DA and 0.2 V for UA, respectively. The detection limits for individual detection and simultaneous detection (S/N = 3) are 0.93 μM and 1.46 μM for AA, 0.11 μM and 0.15 μM for DA, and 0.74 μM and 0.29 μM for UA. The method was applied to the quantitative analysis of AA, DA, and UA in spiked serum samples with satisfying results. Graphical abstract In-situ growth of gold nanoparticles on 3D-networked MoS/rGO nanocomposite for individual and simultaneous determination of ascorbic acid, dopamine and uric acid.