Liquid biopsy analysis represents a suitable alternative analysis procedure in several cases where no tumor tissue is available or in poor patient conditions. Amino acids can play a crucial role in aiding cancer diagnosis. Monitoring of tryptophan (Trp) catabolism can aid in tracking cancer progression. Therefore, a novel nanocomposite was fabricated using overoxidized polypyrrole film doped with nano-carbon dots (nano-CDs) on the pencil graphite electrode (PGE) surface for sensitive evaluation of Trp in human serum. Using square wave voltammetry (SWV), the overoxidized polypyrrole/carbon dots/pencil graphite electrode (Ov-Ox PPy/CDs/PGE) achieved excellent electrochemical catalytic activity for evaluating Trp. The modified electrode, known as Ov-Ox PPy/CDs/PGE, demonstrated superior electrochemical catalytic activity compared to bare PGE, CDs/PGE, PPy/PGE, and PPy/CDs/PGE for evaluation of Trp. The method’s excellent sensitivity was confirmed by the low limits of detection (LOD = 0.003 μmol L-1) and limit of quantitation (LOQ = 0.009 μmol L-1). The biosensor that was developed can measure tryptophan (Trp) levels in the serum of both healthy individuals and female breast cancer patients with high accuracy and sensitivity. The results indicate that there is a significant difference, as shown by the F-test, between healthy individuals and those with breast cancer. This suggests that Trp amino acid could be an essential biomarker
for cancer diagnosis. Consequently, liquid biopsy analysis presents a valuable opportunity for early disease detection, particularly for cancer.