We developed and evaluated two-level, namely 2017011 and 2017012, serum-based reference materials (RMs) for 17 beta-estradiol (17 beta-E2) by the reference method of isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) from the remaining serum samples after routine clinical tests, to help improve clinical routine testing and provide the traceability of results. This paper describes the development process of these RMs. The National Metrology Institute of Japan (NMIJ) certified reference material (CRM) 6004-a was used as the primary RM for the measurement of 17 beta-E2. These serum-based RMs showed satisfactory homogeneity and stability. They also assessed the commutability between the reference method and the three routine clinical immunoassay systems. Besides, a collaborative study was carried out in five reference laboratories, all of which had been accredited by the China National Accreditation Service for Conformity Assessment (CNAS) in accordance with ISO/ WD 15725-1. Statistical analysis of raw results and uncertainty assessment obtained certified values: 2017011 was 445.2 +/- 39.0 pmol/L, and 2017012 was 761.9 +/- 35.5 pmol/L.