Abstract: Copper (Cu) is a crucial trace metal element in human body, which plays an important role in many life processes. Meanwhile, copper is strictly regulated in the human body and its homeostasis is closely related to the physiological state. The occurrences of many diseases are often accompanied by the copper imbalance. By monitoring the subtle variation in copper homeostasis, it can more accurately identify the factors that lead to abnormal changes in human physiology, especially the impact of environmental factors on the occurrence and development of diseases. In the past two decades, with the development of analytical instruments and improvement of analytical methods, non-traditional stable isotopic analysis has been widely applied in geochemistry and archaeology. Owing to its strong traceability, non-traditional stable isotopic analysis can also provide a new perspective to reveal the mechanisms of element homeostasis imbalance. Especially, stable copper isotopic analysis has a wide application prospect in environmental health research. Because of the virtues of wide application range and high analytical accuracy, multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS) has effectively promoted the process of copper isotopic analysis. In this paper, we reviewed the mechanisms of non-traditional stable isotope fractionation and analysis methods of Cu isotopes based on MC-ICP-MS, and the basic conception of mass dependent fractionation (MDF) and non-mass dependent fractionation (MIF) as well as the instrumental configuration of MC-ICP-MS. We also introduced the methods of copper isotopic analysis, and then summarized the main applications of copper isotopic analysis in environmental health research. We focused on two aspects, i.e., Cu isotopic analysis of population characteristics and research on disease indicators. In summarizing the relevant researches of Cu isotopic analysis of population characteristics, we reviewed the influence factors, such as age, gender, dietary habits, and other factors. Afterwards, the studies of copper isotopic analysis on some diseases, such as cancers, nervous diseases, disorders of copper transporters, and other diseases were summarized. Finally, we discussed the application prospects of copper isotopic analysis in environmental health research. Until now, the application of stable copper isotopic analysis in environmental health is still in the initial stage, and there still exist many difficulties and challenges. We primarily list four main difficulties and challenges in future studies, including high-throughput sample analysis, background population investigation, combination of other clinical indicators, and combination of multiple omics techniques.