This paper constructs a water resources security evaluation model from the “man (human activity intensity)-land (geological and geomorphological features)-water (water resources background conditions)” perspective, which accounts for the characteristics of hydrology and water resources in karst areas. A water resources security evaluation index system is established from three aspects (i.e., the background conditions of water resources, human activity intensity and geological and geomorphological features). The evaluation standard threshold is determined in accordance with relevant standards and domestic/foreign development experience. Both the comprehensive weights obtained by the Lagrange coefficient and the multi-objective fuzzy membership functions are used to comprehensively evaluate the spatial and temporal evolution of water resources security in Guizhou Province from 2001 to 2015. The results show that the water resources security comprehensive index of Guizhou Province was between 0.6 and 0.8 during this time (relatively safe type) and the safety was trending upwards. However, the comprehensive index of water resources security of each city in Guizhou Province demonstrates significant spatiotemporal variation. Whilst the comprehensive index was low in the Western part and high in the Eastern part of the province, it generally improved over time for the entire province. Moreover, the criteria layer indexes of water resources security in Guizhou Province and all cities also showed different degrees of variation and regional consistency across space and time. The interaction among these three aspects promoted the spatiotemporal variation of the Water Resources Security Comprehensive Index. In order to safeguard and improve water resources security, it is important to strengthen ecological and environmental management, promote the efficient use of water resources, establish sound management measures and security system related institutions and ensure water resources security.