Objective Protein chromatography is a fundamental technique in protein sciences. Difficulties were always met in experimental teaching for this technique due to its complexity and tedious details. Therefore, we would like to find new strategies for experimental teaching of such technique.Methods We used the 3D simulation techniques to setup an on-line Protein Chromatography Laboratory. We designed the teaching contents, core equipments, the scoring systems, user interface etc. based on our every-day teaching experiences.Results We took the advantage of the 3D simulation techniques, and designed an on-line Protein Chromatography Laboratory exactly reproduced from the equipment-setups of Protein Facility, Zhejiang University School of Medicine. This virtual simulation teaching platform for protein chromatography contains 3 modules regarding the teaching, practicing and on-line exams, so that the efficiency and quality of teaching could be largely improved. In this VR platform for teaching, a new but commonly used type of protein chromatography equipment was chosen in order to complete such shortages in VR teaching for protein purification. Furthermore, fluorescence-detection size-exclusion chromatography (FSEC) and several case studies were also included in the “advance teaching” section.Conclusion Experimental teaching of protein chromatography using virtual simulation was more advantageous than the “old-fashion” ways of teaching. Students could be involved deeply in learning and practicing of such techniques anywhere beside the chromatography laboratory; and based on our teaching experiences, their learning efficiency and quality were indeed improved.