The formation of a protein corona (PC) can significantly impact the detection ability of gold nanoparticles (AuNPs)-small molecule adsorbent probes based on competitive adsorption. To alleviate this problem, in this study, double-stranded DNA (dsDNA) was introduced to modify the AuNP probes to mitigate the negative effect of PCs on the detection of small molecules by taking the AuNPs-dichlorofluorescein (DCF) probe-based detection of ambroxol hydrochloride (AMB) as a study model. It was found that based on the dsDNA-modified AuNPs-DCF probe (dsDNA@AuNPs-DCF), the accuracy for the detection of AMB was significantly improved, which might be attributed to the isolation of proteins from the surface of AuNPs while still allowing small molecules to access the surface due to the introduction of rigid dsDNA. Further, the effect of the strand length and the number of dsDNA modified on the surface of AuNPs on the antifouling performance was then investigated, and it was found that the LOD value of AMB in artificial milk samples by dsDNA