Nanotechnology and nanomaterial based electrochemical biosensors (ECBs) have achieved great development in many fields, such as clinical diagnosis, food analysis, and environmental monitoring. Nowadays, the single-handed pursuit of sensitivity and accuracy cannot meet the demands of detection in many in situ and point-of-care (POC) circumstances. More and more attention has been focused on simplifying the operation procedure and reducing detection time, and thus no-wash assay has become one of the most effective ways for the continuous development of ECBs. However, there are many challenges to realize no-wash detection in the real analysis, such as redox interferences, multiple impurities, non-conducting protein macromolecules, etc. Furthermore, the complex detection circumstance in different application fields makes the realization of no-wash ECBs more complicated and difficult. Thanks to the updated nanotechnology and nanomaterials, in-depth analysis of the obstacles in the detection process and various methods for fabricating no-wash ECBs, most issues have been largely resolved. In this review, we have systematically analyzed the nanomaterial based design strategy of the state-of-the-art no-wash ECBs in the past few years. Following that, we summarized the challenges in the detection process of no-wash ECBs and their applications in different fields. Finally, based on the summary and analysis in this review, we also evaluated and discussed future prospects from the design to the application of ECBs.