Gene expression profiling has become known as a crucial component of horticulture biotechnology, providing essential insights into the genetic and molecular pathways that influence crop yield, quality, and stress resilience. This overview emphasizes the ideas, applications, and integration of gene expression profiling to improve horticulture crops. High-throughput methodologies, including RNA-Seq, microarray analysis, and qRT-PCR, facilitate the discovery of differentially expressed genes associated with critical agronomic properties like as stress tolerance, fruit quality, and disease resistance. Progress in comprehending microRNA regulation, quantitative trait loci (QTL) mapping, and the discovery of molecular markers has expedited marker-assisted selection and genome editing techniques, including CRISPR/Cas9. The integration of transcriptomic data with other omics technologies—metabolomics, proteomics, and genomics—has enabled a systems-level comprehension of numerous plant processes, including secondary metabolite production and postharvest physiology. Although difficulties are associated with data complexity, elevated expenses, and restricted genetic resources in non-model species, novel methods such as single-cell RNA sequencing and artificial intelligence are improving analytical accuracy. Gene expression profiling ultimately connects molecular insights with applied breeding, facilitating the creation of better, climate-resilient, and nutritionally enhanced horticultural cultivars vital for sustainable agriculture.