Aptamer technology has significantly advanced the field of plant research, emerging as a tool for enhancing agricultural productivity, plant growth, and environmental monitoring. Aptamers are short nucleotide or amino acid sequences that can bind to a range of target molecules with high affinity and selectivity. In recent years, these affinity molecules have piqued the interest of researchers across various scientific fields, including pharmaceuticals, analytical chemistry, and plant science. Advancements in aptamer technology have significantly broadened the horizons of plant science, particularly in the areas of plant analyte detection, pathogen targeting, and protein function analysis. Despite the use of various other bioassays and molecular techniques for plant analyte detection, the small size, chemical stability, and cost-effective synthesis of aptamers make them invaluable tools for unravelling the complexities of plant cells. Here, we discuss the progress in the development of nucleic acid and peptide aptamers and summarize their applications in plant biotechnology. The principles and signalling methods of various aptamer-based biosensors and their prospects as biotechnological tools for functional genomic studies, pathogen resistance, and bioimaging are discussed. Finally, the present challenges and future perspectives of aptamer-based technology in plant research are also summarized.