Embodiments of the present disclosure provide a real-time calibration device, a real-time calibration method and a detection apparatus. The real-time calibration device is in fluid communication with a sample injection pipeline of the apparatus to be calibrated. The real-time calibration device is configured to release a trace amount of calibration agent molecules during a sample injection of the apparatus to be calibrated, so that the trace amount of calibration agent molecules and a sample entering the apparatus to be calibrated are mixed and together enter the apparatus to be calibrated, and information of the sample and the calibration agent is detected by the apparatus to be calibrated, thereby performing a calibration.