The development and key technology research of zebrafish online biomonitoring technology can realize real-time monitoring of water quality safety from the perspective of ecosystem by means of 3s technology, and realize early warning of water quality according to the response characteristics of zebrafish. The purpose of this paper is to study the water quality monitoring and early warning technology of zebrafish behavior based on 3s technology. Use GIS technology to process a large number of graphics, data and models, combine graphics and mathematical models, and implement the results of operations on the map. By analyzing the factors that affect the behavior of the indicator biological zebrafish, the results show that these potential physical stress factors have an impact on the normal behavior of zebrafish, but they are also acceptable in the normal range. Zebrafish can adjust and adapt within the fluctuation range of normal water quality, such as pH value and dissolved oxygen, and the behavioral difference is not obvious.