This study presented a HPTLC platformed luminescent biosensor system for screening captan residue. First, the potential bio-effects of layers materials on the detectability of a luminescent bacteria Photobacterium phosphoreum (ATCC 11040) as the sensor cell were assessed. From comparison, it was noteworthy that the combination of sensor cells with normal silica gel layer exclusively gave outstanding detectability (<10 ng/zone). On this basis, HPTLC mediated separation and biosensing was further optimized. Then, the obtained graphic results were digitally quantified via software processing, offering satisfactory selectivity, linearity (R = 0.9901 within 10-80 ng/zone) and sensitivity (0.5 mg/kg against MRLs ≥ 6 mg/kg). Additionally, the performance of the established method was validated with different fruits (recover rates 75-96%, RSD < 11.8%). Meanwhile, it was demonstrated that detectability of this hybrid system would be tuneable by altering the combination of bacteria strains and layer materials, which was meaningful to strengthen the usability of microbial biosensors.