Genetically improved farmed tilapia (GIFT, Oreochromis niloticus) are commercially important fish that are cultured in China. GIFT are highly susceptible to diseases when exposed to high temperatures in summer. Better understanding the GIFT regulatory response to heat stress will not only help in determining the relationship between heat stress signalling pathways and adaption mechanisms, but will also contribute to breeding new high-temperature tolerant strains of GIFT. In this study, we built control (28 °C) and heat-treated (37.5 °C) groups, and extracted RNA from the liver tissues for high-throughput next-generation sequencing to study the miRNA and mRNA expression profiles. We identified 28 differentially expressed (DE) miRNAs and 744 DE mRNAs between the control and heat-treated groups and annotated them using the KEGG database. A total of 38 target genes were predicted for 21 of the DE miRNAs, including 64 negative miRNA-mRNA interactions. We verified 15 DE miRNA-mRNA pairs and 16 other DE mRNAs by quantitative real-time PCR. Important regulatory pathways involved in the early response of GIFT to heat stress included organism system, metabolism, and diseases. Our findings will facilitate the understanding of regulatory pathways affected by acute heat stress, which will help to better prevent heat damage to GIFT.