Transcriptome analysis of probiotic bacteria under heat and salt stress conditions can reveal the physiological response of bacterial cells and related mechanisms in similar environments. The whole-genome map of Lactobacillus acidophilus CICC 6074 was determined and assembled by triple sequencing and bioinformatics analysis. Total RNA was extracted from Lactobacillus acidophilus cells after tolerance to high temperature and high salt conditions and RNA sequencing was performed on the Illumina HiSeq×TEN PE150 platform. The and transcriptomic analysis methods were used to analyze gene expression differences. The results showed that the genome of Lactobacillus acidophilus CICC 6074 was a 1 992 024 bp double-stranded circular DNA molecule without plasmid sequences， with a GC content of 34.71%， and a total of 1 864 genes were predicted. In the high-temperature stress group， a total of 513 significant differences were detected， with 305 significantly up-regulated and 208 significantly down-regulated； in the high salt stress group， a total of 161 significant differences were detected， with 81 significantly up-regulated and 80 significantly down-regulated. The results of functional enrichment analysis indicated that Lactobacillus acidophilus might enhance the adaptation of cells to the environment mainly by enhancing the metabolism of organic acids and transcriptional-translational regulatory processes during high-temperature stress， while enhancing the survival ability mainly by enhancing the metabolism of sugar alcohols and enhancing transmembrane transport during high salt stress.