DNAzymes　with　enzymatic　activity　identified　from　random　DNA　pools　by　in　vitro　selection　have　recently　attracted　considerable　attention.　In　this　work,　a　DNAzyme-based　autonomous-motion　(AM)　molecular　machine　is　demonstrated　for　sensitive　simultaneous　imaging　of　different　intracellular　microRNAs　(miRNAs).　The　AM　molecular　machine　consists　of　two　basic　elements,　one　of　which　is　a　target-analogue-embedded　double-stem　hairpin　substrate　(TDHS)　and　the　other　is　a　locking-strand-silenced　DNAzyme　(LSDz).　LSDz　can　be　activated　by　target　miRNA　and　catalytically　cleave　TDHS,　generating　Clv-TDHS　and　releasing　free　target　analogue　capable　of　triggering　the　next　round　of　cleavage　reaction.　As　such,　the　molecular　machine　can　exert　sustainable　autonomous　operation,　producing　an　enhanced　signal.　Because　the　active　target　analogue　comes　from　the　machine　itself　and　offers　cyclical　stimulation　in　a　feedback　manner,　this　target-induced　autonomous　cleavage　circuit　is　termed　a　self-feedback　circuit　(SFC).　The　SFC-based　molecular　machine　can　be　used　to　quantify　miRNA-21　down　to　10　pM　without　interference　from　nontarget　miRNAs,　indicating　a　substantial　improvement　in　assay　performance　compared　with　its　counterpart　system　without　an　SFC　effect.　Moreover,　due　to　the　enzyme-free　process,　the　AM　molecular　machine　is　suitable　for　miRNA　imaging　in　living　cells,　and　the　quantitative　results　are　consistent　with　the　gold　standard　PCR　assay.　More　interestingly,　the　AM　molecular　machine　can　be　used　for　the　simultaneous　fluorescence　imaging　of　several　intracellular　miRNAs,　enabling　the　accurate　discrimination　of　cancerous　cells　(e.g.,　HeLa　and　MCF-7)　from　healthy　cells.　The　SFC-based　autonomous-motion　machine　is　expected　to　be　a　promising　tool　for　the　research　of　molecular　biology　and　early　diagnosis　of　human　diseases.　©　2021　American　Chemical　Society.