This paper presents the construction and characterization of an amperometric immunosensor based on the graphene/gold nanoparticles (AuNPs/GO) nanocomposite for the detection of the bladder cancer biomarker, apolipoprotein A1 (Apo-A1). The morphological analysis of the AuNPs/GO nanocomposite demonstrated an almost spherical shape of AuNPs and the successful coverage of their surface by graphene oxide. An increased G peak and decreased D peak after the association of AuNPs with GO, implied a reduction in graphene defects. The X-ray photoelectron spectroscopy (XPS) indicated a significant decrease in the quantity of oxygen-containing functional groups in the AuNPs/GO nanocomposite, as compared to the original GO. Furthermore, the developed sensor demonstrated commendable sensitivity and selectivity, with a wide linear range for Apo-A1 detection. Importantly, the immunosensor exhibited remarkable stability over a period of 14 days, signifying its potential for practical applications.