In this study, a novel technique for measuring the thickness of electrospun nanofibrous mat based on image analysis techniques is proposed. The thicknesses of electrospun polyacrylonitrile (PAN), polyvinyl alcohol (PVA), and polyurethane (PU) nanofibrous mats are calculated using depth estimation in different views. The images are captured by a fixed scanning electron microscope (SEM) where the mat sample is rotated by 15 degrees, 30 degrees, and 45 degrees angles. By calculating the disparity value (the distance between two corresponding points in two images), the relative depth of images and consequently the thickness of nanofibrous mat are obtained. Furthermore, the thickness of three electrospun mats are measured from the cross-section view of the nanofibrous mat by scanning the electron microscopy. A close agreement between results obtained by this method at low angle views (15 degrees) and the direct thickness measurement obtained from the cross-section view is achieved. Comparison of the average thickness from the direct measurement and the proposed method for different samples exhibits a linear relationship with the high regression coefficient of 0.96. By using the proposed method, the quantitative evaluation of the thickness measurement becomes feasible over the entire surface of electrospun mats.