Embodiments of the present disclosure provide a line detecting tool and method for a light emitting diode substrate. The line detecting tool for a light emitting diode substrate includes a detecting light source and a liquid crystal plate. The liquid crystal plate is configured to be arranged between the detecting light source and a light emitting diode substrate to be detected and to be in contact with each set of positive electrode and negative electrode of the light emitting diode substrate, so as to transform from a light transmission state to a light shielding state or from the light shielding state to the light transmission state after energizing the each set of positive electrode and negative electrode of the light emitting diode substrate to be detected. The detecting light source is configured to illuminate the liquid crystal plate.