N6-Methyladenosine (m6A) is the most pervasive and evolutionarily conserved epitranscriptomic modification in long noncoding RNA (lncRNA), and its dysregulation may induce aberrant transcription and translation programs. Herein, we demonstrate the methylation-powered assembly of a single quantum dot (QD)-based fluorescence resonance energy transfer (FRET) nanosensor for antibody- and enzyme-free monitoring of locus-specific m6A in clinical tissues. The m6A-sensitive DNAzyme VMC10 is employed to identify a specific m6A site in lncRNA, and it catalyzes the hydrolytic cleavage of unmethylated lncRNA. The cleaved lncRNA fails to trigger the subsequent catalytic hairpin assembly (CHA) reaction due to the energy barrier. In contrast, when m6A-lncRNA is present, the methyl group in m6A protects lncRNA from VMC10-mediated cleavage. With the aid of an assistant probe, the retained intact m6A-lncRNA is released from the VMC10/lncRNA complex and subsequently triggers the CHA reaction, generating abundant AF647/biotin dual-labeled duplexes. The assembly of AF647/biotin dual-labeled duplexes onto 605QD results in efficient FRET between 605QD and AF647. The FRET signal can be simply quantified by single-molecule detection. Notably, this assay can be implemented in an antibody-free and enzyme-free manner. This nanosensor can sensitively quantify target m6A with a detection limit of 0.47 fM, and it can discriminate as low as a 0.001% m6A level from excess coexisting counterparts. Importantly, this nanosensor can monitor the cellular m6A level with single-cell sensitivity and profile target m6A expression in breast cancer and healthy para-cancerous tissues, providing a powerful tool for studying the physiological and pathological functions of m6A.