The activated carbon from marigold flowers (MG) was used to make an unlabeled electrochemical immunosensor to determine prostate cancer. MG was synthesized by hydrothermal carbonization and pyrolysis. MG had a large surface area, was highly conductive, and biocompatible. MG modified with graphene quantum dots produced excellent electron transfer for grafting poly(thionine) (PTH). The amine group of PTH bonded with anti-prostate-specific antigen (Anti-PSA) via glutaraldehyde, forming a layer that improved electron transfer. The binding affinity of the immunosensor, presented as the dissociation constant (Kd), was calculated using the Langmuir isotherm model. The results showed that a lower Kd value indicated greater antibody affinity. The immunosensor exhibited two different linear ranges under optimized conditions: 0.0125 to 1.0 ng mL−1 and 1.0 to 80.0 ng mL−1. The sensor could detect concentrations as low as 0.005 ng mL−1, and had a quantification limit of 0.017 ng mL−1. This immunosensor accurately quantified PSA levels of human serum, and the results were validated using enzyme-linked fluorescence assay (ELFA).