Spider dragline silk is a remarkable material that shows excellent mechanical properties, diverse applications, biocompatibility and biodegradability. Transgenic silkworm technology was used to obtain four types of chimeric silkworm/spider (termed composite) silk fibres, including different lengths of recombinant Major ampullate Spidroin1 (re-MaSp1) or recombinant Major ampullate Spidroin2 (re-MaSp2) from the black widow spider, Latrodectus hesperus. The results showed that the overall mechanical properties of composite silk fibres improved as the re-MaSp1 chain length increased, and there were significant linear relationships between the mechanical properties and the re-MaSp1 chain length (p < 0.01). Additionally, a stronger tensile strength was observed for the composite silk fibres that included re-MaSp1, which only contained one type of repetitive motif, (GA)n/An, to provide tensile strength, compared with the silk fibres that includedre-MaSp2, which has the same protein chain length as re-MaSp1 but contains multiple types of repetitive motifs, GPGXX and (GA)n/An. Therefore, the results indicated that the nature of various repetitive motifs in the primary structure played an important role in imparting excellent mechanical properties to the protein-based silk fibres. A silk protein with a single type of repetitive motif and sufficiently long chains was determined to be an additional indispensable factor. Thus, this study forms a foundation for designing and optimizing the structure of re-silk protein using a heterologous expression system.