Peroxisomes are highly heterogeneous organelles that exhibit remarkable functional diversity across different species and even within different tissues of the same organism. Comprehensive proteomic studies that investigate complex biological roles and regulatory mechanisms depend on the isolation of peroxisomes. Here, we discover that the N-terminal region of cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6α) interacts with the peroxisomal membrane protein ATP-binding cassette sub-family D member 1 & 3 (ABCD1, ABCD3) directly in vitro, and develop an affinity-based strategy PerCap using the specific interactions between ATF6α and ABCDs to rapidly purify native peroxisomes. Using this approach, we isolated peroxisomes from human cells, validated their intactness, and identified the potential peroxisomal proteins by proteomic analysis. We show mitochondrial protein A-kinase anchor protein 1 (AKAP1), Carnitine O-palmitoyltransferase 1 (CPT1A) and NADH-cytochrome b5 reductase 1 (CYB5R1) also localize to peroxisomes, and the loss of AKAP1 affects peroxisome morphology. We also isolated peroxisomes from mouse tissues to compare tissue-specific peroxisomal proteins. Furthermore, by monitoring peroxisomal proteome dynamics during Torin1-induced pexophagy, we identified ubiquitin-conjugating enzyme E2 variant 1 (UBE2V1) as a contributor to this process. In summary, we developed a peroxisome isolation method by discovering the direct interactions between ATF6α and peroxisomal proteins, and established PerCap as a strategy for isolating native peroxisomes from human cells and mouse tissues for proteomic and functional analyses.