In this work, six bovine bone gelatin (type B) samples with varying molecular weight (MW) fractions, comprising α-chains, high- and low-MW fractions, were prepared using ethanol precipitation and pH adjustment. The influence of molecular weight distribution (MWD) on gelatin gel strength was examined, along with the effects of these different MW fractions on microbial transglutaminase (MTGase) cross-linking gelatin. The results showed that, without MTGase treatment, high-MW fractions acted as key fillers in the formation of gelatin gel networks, while α-chains and their aggregates played a central role. In contrast, the gelation ability of low-MW fractions was negligible. MTGase cross-linking significantly increased gel strength in both high- and low-MW samples. For instance, the gel strength of the high-MW sample enhanced from 874.4 g to 1425.9 g, while that of the low-MW sample rose from 186.4 g to 340.4 g. However, gelatin with an MWD featuring fewer high- and low-MW fractions and a higher proportion of α-chains and their aggregates, exhibited a significant decrease in gel strength, which declined from 740.8 g to 560.0 g. These findings emphasized the distinct impact of MW fractions on enzymatic gelation.