The invention discloses a comprehensive resistance weight and graph theory-based river network connectivity assessment method. The method includes the following steps that: a river network is generalized into a directed river network graph model which adopts river sections as edges and adopts the sources, convergences and intersections of rivers, as well as gate dams as vertexes; the resistance items, namely, hydrology, hydrodynamic force, water quality and human activities of any of river sections are calculated, the comprehensive resistance weight (CRW) of the river section is calculated; and the CRW of each river section in the river network is calculated as the previous step, and the CRWs of the river sections are adopted as the weights of the edges, so that a river network weighted adjacency matrix can be established, and a minimum comprehensive resistance matrix can be also obtained, and therefore, the minimum comprehensive resistance of the river network can be obtained, and the reciprocal of the minimum comprehensive resistance of the river network is solved, so that the comprehensive connectivity index (CCI) of the river network can be obtained. According to a method for optimizing a river network gate dam system by using the above method, any one gate dam in the river network is removed sequentially, and comprehensive connectivity indexes (CCI) are calculated again, so that the obtained comprehensive connectivity indexes (CCI) are arranged according to a large-to-small order, and the arrangement order is a priority removing order, and the river network gate dam system is optimized according to the priority removing order.