The release of chemicals and microorganisms from various sources, such as industry, agriculture, animal farming, wastewater treatment plants, and flooding, into water systems have caused water pollution in several parts of our world, endangering aquatic ecosystems and individual health. World Health Organization (WHO) has introduced strict standards for the maximum concentration limits for nutrients and chemicals in drinking water, surface water, and groundwater. It is crucial to have rapid, sensitive, and reliable analytical detection systems to monitor the pollution level regularly and meet the standard limit. Electrochemical biosensors are advantageous analytical devices or tools that convert a bio-signal by biorecognition elements into a significant electrical response. Thanks to the micro/nano fabrication techniques, electrochemical biosensors for sensitive, continuous, and real-time detection have attracted increasing attention among researchers and users worldwide. These devices take advantage of easy operation, portability, and rapid response. They can also be miniaturized, have a long-life span and a quick response time, and possess high sensitivity and selectivity and can be considered as portable biosensing assays. They are of special importance due to their great advantages such as affordability, simplicity, portability, and ability to detect at on-site. This review paper is concerned with the basic concepts of electrochemical biosensors and their applications in various water quality monitoring, such as inorganic chemicals, nutrients, microorganisms’ pollution, and organic pollutants, especially for developing real-time/online detection systems. The basic concepts of electrochemical biosensors, different surface modification techniques, bio-recognition elements (BRE), detection methods, and specific real-time water quality monitoring applications are reviewed thoroughly in this article.