An operating method of a biological detection calibration system, adapted for a biological detection device which includes a light source for emitting a light to detect a biological sample. A step is performed to detect whether a light calibration device exists on the biological detection device, wherein the light calibration device includes a carrier and a calibration sample disposed on the carrier, and the calibration sample includes a light detector. If the light calibration device exists, the intensity of the light detected by the light detector is read to determine whether the intensity of the light detected by the light detector is less than a predetermined value. If the intensity of the light detected by the light detector is less than the predetermined value, a driving voltage of the light source is enhanced, such that the intensity of the light detected by the light detector reaches the predetermined value.