Sensitive, accurate, and nondestructive probing of endogenous messenger RNA (mRNA) in living cells places extremely high demands on nanocarriers and probes and is still a challenge. In the present study, we describe a target-triggered self-assembled DNA tree for amplified analysis of mRNA in intact living cells. The probes assembled into a DNA tree are transported into cells by exosomes, which is beneficial for reducing cell damage and realizing nondestructive analysis. The probes are l-configured single-stranded DNAs (LDNAs) that can resist the degradation of exonuclease and endonuclease, thus laying the foundation for accurate analysis. Under the induction of the target mRNA, the probes in the cells assemble into a small plantlet and eventually grow into a tree after a few rounds of self-cycling, achieving the exponential amplification of fluorescence signals. Compared with the signal amplification based on one-dimensional DNA trunk self-assembly, the three-dimensional DNA tree shows an excellent sensitivity both ex situ and in situ. In this way, favorable sensitivity, accuracy, and nondestructive analysis are integrated into one system. This DNA tree expands the analysis platform for analyzing more biomarkers on a genetic level in an intracellular, nondestructive, and hypersensitive manner and holds great potential in clinical diagnostic and research applications.