Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tuberculosis) is a fatal infectious disease to human health, and the drug tolerance and immune evasion of M. tuberculosis were reported to be related to its biofilm formation; however, the difficulty of M. tuberculosis biofilm culture and its unknown global mechanism impede its further research. Here, we developed a modified in vitro M. tuberculosis biofilm model with shorter culture time. Then we used Illumina RNA-seq technology to determine the global gene expression profile of M. tuberculosis H37Rv biofilms. Over 437 genes are expressed at significantly different levels in biofilm cells than in planktonic cells; among them, 153 were downregulated and 284 were upregulated. Go enrichment analysis and KEGG pathway analysis showed that genes involved in biosynthesis and metabolism of sulfur metabolism, steroid degradation, atrazine degradation, mammalian cell entry protein complex, etc. are involved in M. tuberculosis biofilm cells. Especially, ATP-binding cassette (ABC) transporters Rv1217c and Rv1218c were significantly upregulated in biofilm, whereas efflux pump inhibitors (EPIs) piperine and 1-(1-naphthylmethyl)-piperazine (NMP) inhibited biofilm formation and the expression of the Rv1217c and Rv1218c genes in a concentration-dependent manner, respectively, indicating Rv1217c and Rv1218c are potential target genes of M. tuberculosis biofilm. This study is the first RNA-Seq-based transcriptome profiling of M. tuberculosis biofilms and provides insights into a potential strategy for M. tuberculosis biofilm inhibition.