DNA　walkers,　a　type　of　dynamic　nanomachines,　have　become　the　subject　of　burgeoning　research　in　the　field　of　biology.　These　walkers　are　powered　by　driving　forces　based　on　strand　displacement　reactions,　protein　enzyme/DNAzyme　reactions　and　conformational　transitions.　With　the　unique　properties　of　high　directionality,　flexibility　and　efficiency,　DNA　walkers　move　progressively　and　autonomously　along　multiple　dimensional　tracks,　offering　abundant　and　promising　applications　in　biosensing,　material　assembly　and　synthesis,　and　early　cancer　diagnosis.　Notably,　DNA　walkers　identified　as　signal　amplifiers　can　be　combined　with　various　amplification　approaches　to　enhance　signal　transduction　and　amplify　biosensor　sensing　signals.　Herein,　we　systematically　and　comprehensively　review　the　walking　principles　of　various　DNA　walkers　and　the　recent　progress　on　multiple　dimensional　tracks　by　presenting　representative　examples　and　an　insightful　discussion.　We　also　summarized　and　categorized　the　diverse　signal　amplification　strategies　with　which　DNA　walkers　have　coupled.　Finally,　we　outline　the　challenges　and　future　trends　of　DNA　walker　machines　in　emerging　analytical　fields.　©　2021　Elsevier　B.V.