In this paper, holey nitrogen-doped graphene aerogel (HNGA) was synthesized and applied to the concurrently electrochemical determination of small biological molecules including ascorbic acid (AA), dopamine (DA) and uric acid (UA). Firstly, holey graphene hydrogel was synthesized by the hydrothermal reaction in the presence of HO, which subsequently was lyophilized and further annealed in the mixed gas of ammonia and argon to obtain HNGA. Electron microscopy characterization exhibited a great number of nanopores formed on the basal surface of graphene sheets, and HNGA possessed a hierarchically porous structure. The unique structure and composition of HNGA make it an ideal material for electroanalytical application through accelerating mass and electron transfer. HNGA modified glassy carbon electrode (HNGA/GCE) displayed significantly enhanced electrochemical response to AA, DA, and UA, namely reducing overpotential, increasing current density, and improving the reversibility. The oxidation peaks of these three biomolecules can be entirely separated with evident peak potential differences which are 0.216 V (AA-DA), 0.120 V (DA-UA), and 0.336 V (AA-UA), which it allowed the determination of the three substances at the same time. This sensor shows high sensitivity for the determination of AA, DA, and UA with the detection limit of 16.7 μM, 0.22 μM, and 0.12 μM (S/N = 3), respectively. The proposed sensor was applicable for the practical sample analysis as well and desirable recovery was obtained.