Point-of-care testing (POCT) of tumor markers, such as alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA), can be used for the early diagnosis of cancer. In this paper, a highly sensitive electrochemical immuno-biochip based on a porous three-dimensional graphene aerogel (3D-GA) is presented to detect multiple tumor biomarkers and exosomes. The 3D-GA was prepared via in situ chemical reduction of graphene oxide with L-ascorbic acid and then dehydration by freeze-drying. The obtained 3D-GA exhibits a large specific surface area of 125.3 m2 g-1 due to its intrinsic 3D porous architecture. After chemical activation and modification of the 3D-GA, the prepared microfluidic biochip can be used for detecting various tumor markers in liquid samples via electrochemical impedance spectroscopy (EIS). The electrochemical platform with only 5 μL sample achieved a broad detection range of 1.0 × 10-8-1.0 × 10-5 and 1.0 × 10-8-5.0 × 10-4 mg mL-1 for AFP and CEA, respectively, and a low limit of detection (LOD) of 7.9 and 6.2 pg mL-1 for AFP and CEA respectively, which was much better than the outcomes of many other reports. Moreover, the biochip determined the tumor cell-derived exosomes with a low LOD of 10 particles per μL in the PBS solution and an average recovery rate of ∼90% in the diluted serum.