A brightness calibration method used in an optical detection system includes a single source illuminator and a probe card. The single source illuminator is configured to illuminate the probe card. The probe card has a plurality of detection sites. The brightness calibration method includes: sequentially detecting brightness values at the detection sites through one of a plurality of diffusers by a sensing chip; sequentially detecting transparencies of the diffusers at one of the detection sites by the sensing chip; and selecting and respectively disposing the diffusers corresponding to larger ones of the transparencies over the detection sites corresponding to smaller ones of the brightness values, and selecting and respectively disposing the diffusers corresponding to smaller ones of the transparencies over the detection sites corresponding to larger ones of the brightness values.