The segmentation of the dermal-epidermal junction (DEJ) in in vivo confocal images represents a challenging task due to uncertainty in visual labeling and complex dependencies between skin layers. We propose a method to segment the DEJ surface, which combines random forest classification with spatial regularization based on a three-dimensional conditional random field (CRF) to improve the classification robustness. The CRF regularization introduces spatial constraints consistent with skin anatomy and its biological behavior. We propose to specify the interaction potentials between pixels according to their depth and their relative position to each other to model skin biological properties. The proposed approach adds regularity to the classification by prohibiting inconsistent transitions between skin layers. As a result, it improves the sensitivity and specificity of the classification results.