The invention discloses a light calibration device, a biological detection calibration system and an operating method thereof. The biological detection calibration system includes a biological detection device and a light calibration device. The biological detection device is used for detecting a biological sample and includes a light source and a controller. The light source emits a light to the biological sample. The controller is electrically connected to the light source to drive and control the light source to emit the light. The light calibration device includes a carrier and a calibration sample, wherein the carrier is disposed at a detecting position of the biological sample in the biological detection device to receive the light emitted from the light source. The calibration sample is disposed on the carrier and includes a light detector. The light detector is adapted for detecting the intensity of the light emitted to the carrier and transmitting intensity information to the controller. The controller is adapted for reading the intensity information of the light and comparing the intensity information with a predetermined value to adjust driving energy of the light source, so that the intensity of the light detected by the light detector is not less than the predetermined value.