In tissue engineering, the ideal scaffold should effectively react with cells; this reaction includes cells adhesion, growth, migration and differentiation. Surface properties of scaffold play an important role in the reaction between cells and scaffold. Surface roughness in tissue engineering is one of the surface properties which have some effects on cell behavior. According to the type of cells, the role played by surface roughness or smoothness of scaffold in growth, adhesion, propagation and differentiation is so important. For example for growth and adhesion, bone cells prefer rough surfaces while fibroblast cells prefer smooth surface. In this study, it has been tried to measure nanofiber web surface roughness by using digital signal processing method. Nylon 66, polyacrylonitrile, polyvinyl alcohol and poly acetate polymers were used. Nanofiber webs were produced under optimum conditions, AFM and SEM images of these webs were taken. Because of the presence of noises in the signals, two different algorithms were programmed in order to enhance the surface profile. In the next step, a surface profile was obtained from SEM image of nanofiber web. After that the profile was transformed into two dimensional signals in MATLAB™ software. It was then tried to improve the surface profile and also filtering noises of the signals. Thereafter some of the surface roughness parameters were measured by using signal characteristics terms according to ISO 4287(1997), ASME46, DIN4768. Surface roughness of nanofiber webs was also measured by DME-SPM software and the results of two different methods were compared. According to the results obtained, it can be concluded that the nonlinear filtering method gives acceptable and appropriate results in comparison to the surface roughness values that resulted from AFM. Also nonlinear filtering method was suggested for presenting the roughness properties due to its simplicity and short processing time rather than Bezier function. Due to the more accuracy and simplicity of this method in comparison to the method of using the AFM for soft and thin materials like fibers, the digital signal processing method become more effective for measuring the surface roughness of nanofiber webs. In addition, this method also has the advantage of being a non-contact technique. Keywords: surface roughness, digital signal processing, nanofiber scaffold, electrospinning, atomic force microscope.