Light penetration depth, as a characteristic parameter reflecting light attenuation and transmission in biological tissues, has been applied in nondestructive detection of fruits and vegetables. Recently, with emergence of new optical detection technologies, researchers have begun to explore methods evaluating optical properties of double-layer or even multilayer fruit and vegetable tissues due to the differences between peel and pulp in the chemical composition and physical properties, which has gradually promoted studies on light penetration depth. A series of demonstrated research on light penetration depth could ensure the accuracy of the optical information obtained from each layer of tissue, which is beneficial to enhance detection accuracy for quality assessment of fruits and vegetables. Therefore, the aim of this review is to give detailed outlines about the theory and principle of light penetration depth based on several emerging optical detection technologies and to focus primarily on its applications in the field of quality evaluation of fruits and vegetables, its future applicability in fruits and vegetables and the challenges it may face in the future.