Globally, heavy metal ion (HMI) contamination is on the rise, posing an ever-increasing risk to ecological and human health. In recent years, great research effort has been devoted to the sensitive detection and quantitative analysis of HMIs. Low cost, sensitive, selective, and rapid methods for HMI detection are of growing demand, and HMI biosensors have great potential in meeting this need due to their timeliness, cost-effectiveness and convenience in operation. Glutathione is known for its strong ability to bind with toxic heavy metal ions, in addition to its water solubility, stable activity and ready availability. As a result, glutathione is becoming a molecular probe of choice in the preparation of sensors for sensitive, affordable, and accessible HMI detection. This review summarizes the results from various glutathione-based HMI detection strategies reported in recent years, which are categorized according to their signal transduction methods. Their operation and implementation, along with figures of merit such as limit of detection, selectivity, and response time, are discussed and compared. Based on the review, both individual HMI detection and simultaneous detection of multiple HMIs can be realized under specific reaction conditions, showing the great potential of glutathione-based detection to realize various types of practical HMI detection.