Aflatoxin B1 (AFB1) is a widely distributed contaminant in moldy corn, rice, soybean, and oil crops. Many studies have revealed its adverse effects, such as carcinogenicity, immunotoxicity, and hepatotoxicity, on the health of humans and animals. To investigate the immunotoxic effects on chicken immune organs induced by AFB1, we integrated RNA and small-RNA sequencing data of the spleen and the bursa of Fabricius to elucidate the response of the differentially expressed transcriptional profiles and related pathways. AFB1 consumption negatively influenced egg quality, but no obvious organ damage was observed compared to that of the control group. We identified 3918 upregulated and 2415 downregulated genes in the spleen and 231 upregulated and 65 downregulated genes in the bursa of Fabricius. We confirmed that several core genes related to immune and metabolic pathways were activated by AFB1. Furthermore, 42 and 19 differentially expressed miRNAs were found in the spleen and the bursa of Fabricius, respectively. Differentially expressed genes and target genes of differentially expressed miRNAs were mainly associated with cancer progression and immune response. The predicted mRNA–miRNA pathway network illustrated the potential regulatory mechanisms. The present study identified the transcriptional profiles and revealed potential mRNA–miRNA pathway crosstalk. This genetic regulatory network will facilitate the understanding of the immunotoxicity mechanisms of chicken immune organs induced by high concentrations of AFB1.