Colloidal semiconductor nanocrystals (NCs) have been developed for decades and are widely applied in biosensing/imaging. However, their biosensing/imaging applications are mainly based on luminescence-intensity measurement, which suffers from autofluorescence in complex biological samples and thus limits the biosensing/imaging sensitivities. It is expected for these NCs to be further developed to gain luminescence features that can overcome sample autofluorescence. On the other hand, time-resolved luminescence measurement utilizing long-lived-luminescence probes is an efficient technique to eliminate short-lived autofluorescence of samples while recording time-resolved luminescence of the probes for signal measurement after pulsed excitation from a light source. Despite time-resolved measurement being very sensitive, the optical limitations of many of the current long-lived-luminescence probes cause time-resolved measurement to be generally performed in laboratories with bulky and costly instruments. In order to apply highly sensitive time-resolved measurement for in-field or point-of-care (POC) testing, it is essential to develop probes possessing high brightness, low-energy (visible-light) excitation, and long lifetimes of up to milliseconds. Such desired optical features can significantly simplify the design criteria of time-resolved measurement instruments and facilitate the development of low-cost, compact, sensitive instruments for in-field or POC testing. Mn-doped NCs have recently been in rapid development and provide a strategy to solve the challenges faced by both colloidal semiconductor NCs and time-resolved luminescence measurement. In this review, we outline the major achievements in the development of Mn-doped binary and multinary NCs, with emphasis on their synthesis approaches and luminescence mechanisms. Specifically, we demonstrate how researchers approached these obstacles to achieve the aforementioned desired optical properties on the basis of the progressive understanding of Mn emission mechanisms. Afterward, we review representative applications of Mn-doped NCs in time-resolved luminescence biosensing/imaging and present the potential of Mn-doped NCs in advancing time-resolved luminescence biosensing/imaging for in-field or POC testing.