The development of active and defective sites containing mesoporous carbon material is highly crucial for high-performance sensors. In this work, N-doped hollow carbon sphere (NHCS) materials with high surface area and large volume of graphitic and pyridinic N were obtained by variation of the carbonization temperature between 700–1000 °C. Here, dopamine hydrochloride and tetraethyl orthosilicate (TEOS) were used as C, N and silica precursors, with both involved in silica-poly (dopamine) (PDA) sphere formation followed by silica etching to NHCS. Among NHCS materials, NHCS-1000 exhibited an improved surface area, porosity, layer thickness, defective sites and graphitic along with pyridinic sites. An NHCS-modified glassy carbon electrode (NHCS/GCE) was fabricated and successfully used for the simultaneous sensing of ascorbic acid (AA), uric acid (UA), dopamine (DA) and acetaminophen (AC). All NHCS materials exhibited electrocatalytic activity towards the oxidation of these four biologically important analytes. Compared with other carbon materials, NHCS-1000 showed a higher electrocatalytic activity. Hence, the NHCS-1000-modified electrode was used for the further electrochemical sensing of multiple analytes. The limit of detection (LOD) values were 0.135, 0.012, 0.131 and 0.095 μM, respectively, for AA, UA, DA and AC. From the calibration plot, the calculated sensitivity values were: 0.029 mA cm⁻² mM⁻¹ for AA, 0.73 mA cm⁻² mM⁻¹ for DA, (0.53; 0.069 mA cm⁻² mM⁻¹) for UA, and (0.53; 0.765 mA cm⁻² mM⁻¹) for AC. The linear ranges of AA, DA, UA and AC were 0.01–7.0 mM, 0.001–1.5 mM, (0.005–1.0; 2.0–6.0 mM), and (0.05–1.0; 2.0–6.0 mM), respectively. The NHCS-1000-modified electrode showed good selectivity and linear range. The proposed NHCS-1000/GCE was effectively used for the detection of multiple analytes in the real samples of urine, serum, ascorbic acid and commercially available tablets.