Lower reaches of the Amu Darya River Basin (LADB) is one of the typical regions which is facing the problem of water shortage in Central Asia. During the past decades, water resources demand far exceeds that supplied by the mainstream of the Amu Darya River, and has resulted in a continuous decrease in the amount of water flowing into the Aral Sea. Clarifying the dynamic relationship between the water supply and demand is important for the optimal allocation and sustainable management of regional water resources. In this study, the relationship and its variations between the water supply and demand in the LADB from the 1970s to 2010s were analyzed by detailed calculation of multi-users water demand and multi-sources water supply, and the water scarcity indices were used for evaluating the status of water resources utilization. The results indicated that (1) during the past 50 years, the average total water supply (TWS) was 271.88 × 108 m3/y, and the average total water demand (TWD) was 467.85 × 108 m3/y; both the volume of water supply and demand was decreased in the LADB, with rates of −1.87 × 108 m3/y and −15.59 × 108 m3/y. (2) percentages of the rainfall in TWS were increased due to the decrease of inflow from the Amu Darya River; percentage of agriculture water demand was increased obviously, from 11.04% in the 1970s to 44.34% in 2010s, and the water demand from ecological sector reduced because of the Aral Sea shrinking. (3) the supply and demand of water resources of the LADB were generally in an unbalanced state, and water demand exceeded water supply except in the 2010s; the water scarcity index decreased from 2.69 to 0.94, indicating the status changed from awful to serious water scarcity. A vulnerable balanced state has been reached in the region, and that water shortages remain serious in the future, which requires special attention to the decision-makers of the authority.