Rationale: Atmospheric pressure interface multi‐reflection time‐of‐flight mass spectrometry (API‐MRTOF‐MS) has the potential to be a rapid and high‐resolution analytical tool for versatile applications in chemistry, biology, environmental science, and medicine. Methods: The ions were reflected in a mass analyzer via electrostatic mirrors and folded flight path. Therefore, flight distances were significantly increased. The ion flight path of the API‐MRTOF‐MS was extended from meters to over 1 km, and the mass resolution was increased. Furthermore, the mass analysis could be completed at around 10 ms due to the rapid response of TOF‐MS. Results: A high‐resolution API‐MRTOF‐MS approach is successfully developed in this study. The mass resolution could achieve 116 050 (full widths at half maximum [FWHM]) for Cs+ ions using an atmospheric pressure electrospray ionization within a total TOF of only 18 ms. An ion transmission efficiency of over 50% was achieved after 600 cycles. Conclusions: The analytical performance of the newly developed API‐MRTOF‐MS demonstrated that it is suitable for high resolution and rapid analysis in many fields. [ABSTRACT FROM AUTHOR]