Homologues of the T-box gene Brachyury play an essential role in notochord specification of early embryo development of chordates. Amphioxuses, also called cephalochordates, represent the most basally divergent lineage of chordates, being the sister group of urochordates and vertebrates. It is now generally agreed that amphioxus is the first animal group to evolve a real sense of notochord in all metazoans, hence studying Brachyury in amphioxus would shed important insights into the origin and evolution of notochord. In order to explore the function of Brachyury in the developmental regulation network of amphioxus, we generated a mouse anti-amphioxus BRA polyclonal antibody. Amphioxus possesses two Brachyury homologues genes: Bra1 and Bra2, encoding two proteins with a sequence similarity of 93% (Fig. 1a). It is very hard to distinguish specific epitopes between them. The transcriptome data showed that the expression level of Bra2 was much higher than that of Bra1 (Fig. 1b). Further analysis of BRA2 sequence feature indicated that ideal presume antigenic determinants located in the N-terminal of BRA2 (Fig. 1c). Therefore, a 696 bp gene segment was amplified by PCR from Bra2 N-terminal (Fig. 2a) and inserted into prokaryotic expression vector pET28a (Fig. 2b). The recombinant plasmid pET28a-Bra2-N was transformed into Escherichia coli BL21 and induced to express the tagged protein (Fig. 3a). We obtained a huge amount of soluble recombinant protein with an expected size (31 ku) (Fig. 3b) and purified the tagged protein using Ni2+ affinity chromatography (Fig. 3c). Three ICR mice were immunized to generate polyclonal antibodies against amphioxus BRA2 with purified recombinant protein (1.3 g/L). The enzyme-linked immunosorbent assay (ELISA) showed that the titer of mouse anti-amphioxus BRA2 antibody was 1︰256 000, with a high sensitivity (Fig. 4a). Western blot experiments showed that the polyclonal antibody could not only effectively identify recombinant protein (Fig. 4b) but also recognized amphioxus BRA1 and BRA2 (Fig. 4c, Fig. 5a, b). In conclusion, we successfully generated the mouse anti-amphioxus BRA polyclonal antibody that would be a powerful molecular tool for further investigating the function of Brachyury in amphioxus.