A label-free, versatile and low-background chemiluminescence (CL) sensing strategy based on gold nanocluster catalysis combined with the separation of magnetic beads (MBs) was developed. Kanamycin was selected as the target analyte to exhibit the analytical performance of this platform. Two single-stranded DNA (named DNA1 and DNA2) are ingeniously designed. DNA1, containing an aptamer sequence of the targets, was firstly immobilized on the MBs which were modified with many amino groups by amidation reaction. DNA2 consists of 30 repeat adenosine bases (A₃₀) at the 5′ terminal which were used to prepare AuNCs by a UV-light-assisted method and a 12 nucleotide sequence at the 3′ terminal which can easily hybridize with DNA1 to form a partly complementary double-stranded structure. In the presence of targets, the aptamer modified on MBs would combine with targets and lead to release the prepared DNA-templated AuNCs. After the magnetic separation, enrichment AuNCs in the supernatant can catalyze the CL substrate to produce a strong CL signal. The well-designed CL sensing strategy exhibited a low detection limit of 0.035 nM for kanamycin, and it also showed good selectivity and stability. Most importantly, different targets can be analyzed only by changing the aptamer sequence that is immobilized on the MBs. Therefore, the strategy we proposed here has provided a versatile sensing platform for sensitively detecting various biomolecules at low levels.