An antisense oligonucleotide (ASO) is a short single-stranded deoxyribonucleotide complementary to the sense strand of a selected nucleic acid. As a result, an ASO can modulate gene expression through several mechanisms. The technology based on ASO has already been applied in studies on gene function in mammalian cells and selective therapeutic strategies for many diseases. The conceptual simplicity and low cost of this method, and the developments in the field of plant genome sequencing observed in the last decades, have paved the way for the ASO method also in plant biology. It is applied in gene function analysis as well as the development of non-invasive plant production technology involving gene modifications without transgenesis. Therefore, the first part of this review provides a comprehensive overview of the structure, mechanism of action and delivery methods of ASOs in plants and shows the most important features essential for the proper design of individual experiments. We also discuss potential issues and difficulties that may arise during practical ASO implementation. The second part of this article contains an analysis of ASO applications in various studies in the field of plant biology. We presented for the first time that ASOs were also successfully applied in cucumber.