Despite the integrative function of miRNAs and genes in chicken embryonic gonadal sex differentiation, their roles remain poorly understood. Here, we used RNA-seq to analyze 12 gonadal samples from embryonic days 5 (E5) and 9 (E9). Our analysis of miRNA and mRNA expression during this key developmental stage revealed five DEmiRNAs and 235 DEmRNAs consistently associated with left-right asymmetry in female gonads in both F5L vs F5R and F9L vs F9R comparisons. The interaction between DEmiRNAs and target DEmRNAs was analyzed in relation to the left-right asymmetry of embryonic gonadal development at E5 and E9 in chickens. The KEGG pathway enrichment analysis revealed that several significant pathways were involved. These include the complement and coagulation cascades, tight junctions, and synthesis and secretion of aldosterone. Differentially expressed miRNAs and target genes at embryonic days 5 and 9 reveal key molecular mechanisms driving gonadal sex differentiation in chickens.