Inhibitors of histone deacetylases (HDACs) have been considered to be new anticancer agents. As a key inhibitor of HDAC, vorinostat can cause growth arrest and death of a broad of transformed cells and interact with a variety of substrates. A comprehensive analysis of proteins interacting with HDAC inhibitors is of great importance in understanding molecular mechanisms of the drugs. Here, we reported the preparation and characterization of vorinostat-coated beads for profiling of novel target proteins of vorinostat (a key HDAC inhibitor). The enriched proteins were further analyzed by HPLC-MS/MS. Besides the known substrates, there were also several novel enriched protein candidates, one of which was a metalloenzyme α-enolase (ENO-1). According to our best knowledge, it is the first time that ENO-1 has been detected as a potential target of vorinostat through chemoproteomics approach. Further competition analysis indicated that ENO-1 may be co-enriched as a substrate complex. Our results demonstrated that the chemical probe combined with proteomics approach may be developed as a potential tool to identify target proteins of drugs.