This study described the development of a photocatalytic electrochemical aptasensor for the purpose of detecting chloramphenicol (CAP) antibiotic residues in water by utilizing SYBR Green I (SG) and chemically exfoliated MoS2 (ce-MoS2) as synergistically signal-amplification platforms. The Au nanoparticles (AuNPs) were electrodeposited onto the surface of an indium tin oxide (ITO) electrode. After that, the thiolate-modified cDNA, also known as capture DNA, was combined with the aptamer. Subsequently, photosensitized SG molecules and ce-MoS2 nanomaterial were inserted into the groove of the resultant double-stranded DNA (dsDNA). The activation of the photocatalytic process upon exposure to light resulted in the generation of singlet oxygen. This singlet oxygen effectively splits the double-stranded DNA, resulting in a significant enhancement in the current of [Fe(CN)6]3-/4-. When the CAP was present, both SG molecules and ce-MoS2 broke away from the double-stranded DNA, which turned off the photosensitization response, leading to a significant decrease in the current of [Fe(CN)6]3-/4-. Under optimal conditions, the aptasensor exhibited a linear relationship between CAP and logarithmic concentrations from 20 to 1000 nM, with a detection limit (3σ) of 3.391 nM. The aptasensor also demonstrated good selectivity towards CAP in the presence of interfering antibiotics such as tetracycline, streptomycin, levofloxacin, ciprofloxacin, and sulfadimethoxine. Additionally, the results obtained from the analysis of natural water samples using the proposed aptasensor were consistent with the findings acquired through the use of a liquid chromatograph-mass spectrometer. Therefore, with its simplicity and high sensitivity, this biosensor can potentially detect alternative antibiotics in environmental water samples by replacing the aptamers based on photosensitization.