Reference materials (RMs) are the foundation in isotopic analysis, and the assignment of reference values of isotope ratios in RMs is a complex process. In this study, we established new isotopic reference values and its uncertainties for the Sr, Nd, Hf and Pb isotope ratios in 18 commonly used pure materials and rock RMs, and for Hf and O isotope ratios in five zircon RMs, following the ISO guidelines and procedures in the IAG Certification Protocol. The original data were collected from the geochemical database “GeoReM”. An estimation method for generating reference values of isotope ratios was developed, including a data filter process, technical criteria assessment and robust statistical calculation. The generated analytical results demonstrate the same instrumental performance with both TIMS and MC-ICP-MS for Sr and Nd isotopic analysis. However, significant discrete data for the Pb isotope ratios obtained by TIMS and MC-ICP-MS are evident, indicating existing challenges for accurate Pb isotopic analysis. It was found that 91500 is not a suitable Hf isotopic RM for micro-analysis due to the heterogeneous Hf isotopic composition and the Ple?ovice zircon may be a more ideal alternative. This study provides a powerful protocol and practical examples for estimating reference values of isotope ratios in frequently used RMs.