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OBJECTIVES The aim ofthis study was to analyse the protein profiles of acquired enamel pellicle (AEP) at two time points (5 minutes and 2 hours) between caries-susceptible and caries-free subjects using label-free and parallel reaction monitoring (PRM) proteomic approaches, and to discover potential biomarkers for dental caries. METHODS Sixty participants (30 caries-susceptible (DMFT＞5) and 30 caries-free subjects (DMFT = 0)) were included. Their AEP at 5 minutes and 2 hours was separately sampled, and the AEP materials were quantitatively analysed using label-free proteomics. Bioinformatics analysis of differentially expressed proteins was subsequently conducted and target proteins were verified using PRM analysis. RESULTS A total of 82 and 39 differentially expressed proteins were identified at the 5-minute and 2 -h points, respectively, between the caries-susceptible and caries-free groups. Some proteins related to immune response and antibacterial activity were identified, and the characteristic enriched biological processes of up- and down-regulated proteins were presented using bioinformatics analysis. Subsequently, five intriguing target proteins of each group were chosen for PRM analysis and their presence was successfully verified in the 5-minute and 2 -h AEPs, respectively. CONCLUSIONS The proteins inthe AEPs of caries-susceptible and caries-free subjects at both time points presented unique protein profiles related to immune response and antibacterial activity. Mucin-7 can be regarded as an important potential AEP biomarker for dental caries. CLINICAL SIGNIFICANCE The results of the study identified potential biomarkers, which can facilitate the design of new bio-functional agents for the diagnosis and treatment of dental caries in future.