Genome editing is a powerful genetic approach for studying gene function by disrupting target genes or integrating exogenous genes into the targeted regions of the genome. In insects, highly efficient gene knockouts have been achieved across various species. However, the efficiency of gene knock-in is much lower compared with gene knockout, particularly when a long gene cassette is integrated into the genome by homology-directed repair (HDR) of double-strand breaks (DSBs). An alternative approach for gene knock-in is microhomologymediated end-joining (MMEJ), which uses microhomology to precisely insert exogenous genes into target loci and has been successful for several genes in the silkworm. To further evaluate its efficacy and broader application, we performed MMEJ-knock-in in the silkworm targeting the Fibroin-L (Fib-L) gene. We inserted a whole knock-in donor plasmid (~4.5 kb) carrying an eye-specific fluorescent marker gene (3xP3-DsRed2) into the first exon of Fib-L. Our results indicated that the whole donor plasmid was efficiently and precisely inserted into the target site. Notably, all larvae carrying the precise knock-in allele exhibited red fluorescence in the posterior silk gland, in addition to the eyes, suggesting that the inserted 3xP3-DsRed2 trapped the enhancer of the original Fib-L, which is specifically expressed in the posterior silk gland. Taken together, our results indicate that MMEJ is a promising and feasible approach for efficient and precise gene knock-in in the silkworm.