Self-assembly processes exist widely in life systems and play essential roles in maintaining life activities. It is promising to explore the molecular fundamentals and mechanisms of life systems through artificially constructing self-assembly systems in living cells. As an excellent self-assembly construction material, deoxyribonucleic acid (DNA) has been widely used to achieve the precise construction of self-assembly systems in living cells. This review focuses on the recent progress of DNA-guided intracellular self-assembly. First, the methods of intracellular DNA self-assembly based on the conformational transition of DNA are summarized, including complementary base pairing, the formation of G-quadruplex/i-motif, and the specific recognition of DNA aptamer. Next, The applications of DNA-guided intracellular self-assembly on the detection of intracellular biomolecules and the regulation of cell behaviors are introduced, and the molecular design of DNA in the self-assembly systems is discussed in detail. Ultimately, the challenges and opportunities of DNA-guided intracellular self-assembly are commented.