Background:　Sexually　transmitted　infections　(STIs)　rank　among　the　most　prevalent　acute　infectious　conditions　and　remain　a　major　global　public　health　concern.　Notable　STI　pathogens　include　Chlamydia　trachomatis　(CT),　Ureaplasma　urealyticum　(UU),　and　Neisseria　gonorrhoeae　(NG).　Early　detection　and　diagnosis　are　crucial　for　controlling　the　spread　of　STIs.　Results:　In　this　study,　we　utilized　toehold　switches　integrated　with　a　cell-free　system　to　develop　a　simple,　colorimetric,　sensitive,　specific　and　rapid　method　for　the　parallel　detection　of　CT,　UU,　and　NG.　Target　DNA　and　sensor　DNA　were　transcribed　into　target　trigger　RNA　and　toehold　switch　sensor　RNA　respectively,　within　a　cell-　free　transcription　system.　The　binding　of　target　RNA　to　the　toehold　switch　RNA　activated　the　switch,　subsequently　initiating　the　translation　of　the　downstream　lacZ　gene.　The　expressed　LacZ　protein　hydrolyzed　the　substrate　chlorophenol　red-beta-D-galactopyranoside　(CPRG),　resulting　in　a　color　change　from　yellow　to　purple,　which　provided　a　visible　colorimetric　output.　The　three　screened　sensors　exhibited　excellent　orthogonality　without　any　observed　cross-reactivity.　By　enhancing　sensitivity　with　recombinase　polymerase　amplification　(RPA),　we　reliably　detected　NG　in　clinical　samples　using　this　method,　with　no　interference　from　other　pathogens.　Moreover,　we　selected　high-performance　toehold　switch　sensor　for　paper-based　detection,　further　enhancing　portability.　Significance:　In　summary,　this　technique　enables　the　simple　snd　sensitive　parallel　detection　of　CT,　UU,　and　NG,　generating　visible　colorimetric　results　without　the　need　for　specialized　personnel　or　sophisticated　equipment.　Given　these　advantages,　this　method　holds　significant　potential　as　a　simple　and　portable　diagnostic　tool　in　resource-limited　settings　or　point-of-care　testing　(POCT)　scenarios.