An increasing amount of evidence has revealed that microRNAs (miRNAs) participated in immune regulation and reaction to acute cold and heat stresses. As a new type of post-transcriptional regulatory factor, miRNA has received widespread attention; However, the specific mechanism used for this regulation still needs to be determined. In this study, thirty broilers at the same growth period were divided into three groups and treated with different temperature and humidity of CS (10-15 °C and 90% Relative Humidity (RH)), HS (39 °C and 90% RH), and NS (26 °C and 50-60% RH) respectively. After 6 h, splenic tissues were collected from all study groups. miRNA sequencing was performed to identify the differentially expressed miRNAs (DEMs) between HS, CS, and NS. We found 33, 37, and 7 DEMs in the HS-NS, HS-CS, CS-NS group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that DEMs were significantly enriched in cytokine-cytokine receptor interaction and functioned as the cellular responders to stress. We chose two miRNA, miR-34a and miR-449c, from the same family and differential expressed in HS-CS and HS-NS group, as the research objects to predict and verify the target genes. The dual-luciferase reporter assay and quantitative real-time PCR (qRT-PCR) confirmed that two cytokines, IL-2 and IL-12α, were the direct target genes of miR-34a and miR-449c. To further understand the mediation mechanism of miRNAs in acute cold/heat-stressed broiler chicken, a splenic cytokines profile was constructed. The results showed that IL-1ß was strongly related to acute heat stress in broiler chicken, and from this we predicted that the increased expression of IL-1ß might promote the expression of miR-34a, inducing the upregulation of interferon-γ (INF-γ) and IL-17. Our finds have laid a theoretical foundation for the breeding of poultry resistance and alleviation of the adverse effects of stress.