A calibration strip and a laser calibration system using thereof are disclosed. The calibration strip is comprised of: a substrate; and a light impermissible layer, having a calibration pattern formed thereon while being formed on the substrate. The light impermissible layer is an opaque layer, being formed on the surface of the substrate by coating, electroplating or adhering. The substrate, manufactured by the principle for enabling the color or brightness of the substrate to have high contrast comparing with those of the light impermissible layer, can be a structure of a layer of transparent material and a light source; a layer of transparent material and a backlight source; or a metal film having a reflective layer formed thereon. Since, in the laser calibration system, the calibration strip with the calibration pattern is imaged by an imaging device and then the captured image is send to a processing unit where it is analyzed, the time-consuming and inaccurate off-line manual calibration is no longer required and the laser calibration system can be adapted for various lasers regardless of their spectra.