Abstract: To study the molecular mechanisms of biosurfactant synthesis in Achromobacter sp. HZ01, strain HZ01 was cultured in the media containing citric acid and sodium pyruvate（control） for three days, respectively. The Illumina Hiseq 2500 was employed to perform transcriptome sequencing. After data quality control, sequence alignment,screening of differentially expressed genes and gene annotation were carried out. Fluorogenic quantitative PCR（RT-qPCR） was employed to verify the gene differential expression analysis. The results showed that strain HZ01 could use citric acid and sodium pyruvate as carbon source for growth, respectively. The fermentation broth using citric acid as carbon source exhibits favorable emulsifying activity. The most significantly up-regulated and down-regulated genes encoded OmpW and dioxygenase, respectively. "Amino acid transport and metabolism" contained the largest number of differentially expressed genes in the eggNOG database. “ABC transporters”contained more differentially expressed genes in the KEGG database. RT-qPCR showed that the results of gene differential expression analysis were reliable. In this study, the identified potential genes related to biosurfactant synthesis in strain HZ01 mainly included the genes coding for glycosyltransferase, 3-oxoacyl-ACP reductase, 3-oxoacyl-ACP synthetase, and thioesterase. Glyoxylate cycle may play an important role in the glycolipid synthesis in strain HZ01. This study reveals the potential genes of biosurfactant synthesis in Achromobacter sp. HZ01, laying the foundation for future in-depth studies on the molecular mechanisms of biosurfactant synthesis and construction of engineering bacteria.