Electrochemical biosensors represent a class of sensors that employ biological materials as sensitive elements, electrodes as conversion elements, and potential or current as detection signals. The integration of CRISPR/Cas systems into electrochemical biosensors holds immense potential, offering enhanced versatility, heightened sensitivity and specificity, reduced recovery time, and the ability to capture and identify analytes at low concentrations. In this review, we provided a succinct summary of the fundamental principles underlying electrochemical biosensors and CRISPR/Cas systems, and new progress of electrochemical biosensors based on CRISPR/Cas systems in virus, bacteria, and cancer detections. Besides, we discussed its pros and cons, present gaps, potential problem-solvers, and future prospects. To sum up, CRISPR/Cas mediated electrochemical biosensors will surely benefit us a lot in the detection of cells and microorganisms, and of course in other promising fields.