Ancient living halotolerant bacteria and ancient DNA have been reported from bedded marine halite, but seldom from lacustrine halite of the geological age. A halotolerant bacterium (Haloalkaliphilic bacterium, designated as 3-4) was isolated from primary halite with abundant fluid inclusions. Primary halite crystallized in lacustrine Yunying Depression located in the northeastern Jianghan Basin of central China. Yunying Depression is an inland salt paleolake residing in a faulted basin formed during late Cretaceous/early Tertiary. Brine inclusions from the same layer of the salt that housed Haloalkaliphilic bacterium 3-4 are the middle-late Eocene primary halite crystals and have homogenization temperatures between 5.8 and 43.3 °C. The estimated age is between 33.9 and 48.6 Ma years old for these fluid inclusions, and this represents the long-term survivability of this microorganism Haloalkaliphilic bacterium 3-4. Sequence analysis of the partial fragment of 16S ribosomal DNA showed that the organism is similar to the lineage of Haloalkaliphilic bacterium and Oceanobacillus sp. The isolation of the microbe reinforces the long-term preservation possibility of life in extreme environment such as the fluid inclusions in halite.