The accurate detection of vital biomarkers such as Ascorbic Acid (AA), Uric Acid (UA) and Nitrite ( NO2 −) is crucial for human health surveillance. However, existing methods often struggle with concurrent detection and quantification of multiple species, highlighting the need for a more effective solution. To address this challenge, this study aimed to develop a multifunctional electrochemical sensor capable of parallel detection of AA, UA and NO2 − using a synergistic combination of Graphene Oxide (GO) and Cadmium Sulfide (CdS) materials. Notably, the fabricated CdS@GO/Glassy Carbon Electrode (GCE) exhibited exceptional electrochemical activity, as evidenced by Differential Pulse Voltammetry (DPV) analysis. The sensor demonstrated remarkable sensitivity (8.13, 10.12, and 9.05 μA·μM−1·cm−2) and ultra-low detection limits (0.034, 0.062, and 0.084 μM) for AA, UA and NO2 −, respectively. Furthermore, it successfully identified single molecules of each analyte in aqueous and biologic fluid samples, with recovery values comparable to those obtained using High-Performance Liquid Chromatography (HPLC) standard addition methods. The significance of this study lies in developing a novel CdS@ GO/GCE sensor that enables concurrent detection and quantification of multiple vital biomarkers, offering a promising tool for human health monitoring and diagnosis.