The invention relates to a temperature-controlled microculture instrument and a culture method. The temperature-controlled microculture instrument includes a ventilating fan, a fan cabinet, a ventilating opening, ultraviolet lampshades, a cabinet door, a CCD camera, a radiation lampshade, a signal line, an instrument panel, a control line, a control cabinet, a display screen, control buttons, a charging port, a dirt discharging tube, a limiting block, a temperature and humidity sensor, pulley, a carrying box, an incubator and a guide rail. A using method of the instrument includes the following six steps: maintenance work, sample preparation, sample cultivation, detection work, inactivation treatment and resetting for later use. The instrument has the advantages of simple system formationstructure, high integration and high running automation degree, effective improvement of the flexibility, convenience and working efficiency of microculture work, effective improvement of the workingefficiency and precision of the detection operation, effective avoiding of harms and pollution of a detection sample to an operator and the surrounding environment, and improvement of the safety and reliability of the operation.