The chimeric signal peptides superior to natural signal peptides were constructed based on the N, H and C regions of different natural signal peptides to improve the α-amylase secretion in Bacillus subtilis. Through screening the natural signal peptides library, several optimal natural signal peptides were selected for Bacillus amyloliquefaciens α-amylase secretion in Bsubtilis. The H region of signal peptide SPsacB were replaced by H region of four natural signal peptides (SPyvcE, SPyoqM, SPBglS and SPyobB) to construct four different chimeric signal peptides (RSP1 to RSP4) applied in α-amylase genetic engineering strain. The results show that the most suitable natural and chimeric signal peptides for α-amylase were SPyoqM and RSP1, respectively. The α-amylase secretion efficiency guided by RSP1 was 27% higher than that by SPyoqM, 36 times higher than that by SPyvcE which has the same H region with RSP1, and 207 times higher than that by SPsacB which has the same N and C region but different in H region. The chimeric signal peptide construction strategy is effective for α-amylase secretion in Bacillus subtilis, which provides guidance for efficient secretion of other proteins.