Limonium bicolor, a halophytic species of Plumbaginaceae, can thrive in saline or saline-alkali (sodic) soil, demonstrating that it has developed an efficient saline-alkali resistance system, and is an ideal material for the study of saline-alkali tolerance. In order to identify and characterize the complexity of this adaptation, expressed sequence tags (ESTs) analysis and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were conducted. We constructed a cDNA library of L. bicolor exposed to 0.4M NaHCO3 for 48h, and obtained 2358 ESTs, representing 1735 unique genes. A BLASTX search revealed that 1393 ESTs, representing 873 unique genes, showed significant similarity (E-values <10(-4)) to protein sequences in the non-redundant database. These ESTs were further grouped into 12 functional categories according to their functional annotation. The most abundant categories were metabolism (18.74%), photosynthesis (14.86%), unknown classification (12.20%), defense (12.20%), and transport facilitation (10.19%). In total, 286 putative abiotic stress related transcripts, representing 121 unique genes, were identified. Among them, the two most abundant genes encoded metallothionein (EH794553) and lipid transfer protein (EH794695), each of which accounted for 1.4% of the total ESTs. The expression of 18 putative stress-related genes were further analyzed in roots and leaves of L. bicolor using real-time RT-PCR, and 14 genes were differentially expressed by more than 2-fold as a result of the NaHCO3 stress. The results of this study may contribute to our understanding of the molecular mechanism of saline-alkali tolerance in L. bicolor.