Many studies have been conducted to evaluate seam pucker and to investigate the effect of sewing parameters on seam pucker rate and solutions to reduce this apparent defect in textiles have been proposed. In this study, seam pucker of the fabric in the seam in multi-layer insulation blanket (3 and 7 layers) prepared from two different materials (thin layers covered with aluminum and perforated wrap kintted fabric made of 100% PET) was investigated, and the effect of sewing parameters such as stitch length, needle thread tension, presser foot pressure, rotational frequency of main shaft and needle count on puckering of (MLI) was studied. MLI blanket are used in the telecommunications and aerospace industries to insulate the electronic components of satellites, spacecraft, etc. against the sun's rays in the Earth's atmosphere, so that the device is protected from damage caused by the sun's rays. Due to the distructive effects of seam pucker in insulation, reducing the amount of this phenomenon in multilayer insulation improves the performance of these blanket, which requires the evaluation and analysis of puckers in different layers. In this study a new method was introduced to analyze seam puckering in MLI. Specimens are sewen with lockstitch sewing machine and scanned with laser scanning system. Scanned images are processed via Matlab software and parameters including relative pucker coefficient (k), pucker sharpness, sub layer surface area, length shrinkage were measured. It was observed that the effect of stitch length, needle thread tension and presser foot pressure was more than that of other factors on the seam pucker. None of Rotational frequency of main shaft and needle count alone had any considerable effect on increasing or decreasing the seam pucker and had a different effect when considered another parameter. Also was found when the number of layers increased, relative pucker coefficient in first layer in 7-layer specimen was lower than 3 layer. Overall, relative pucker coefficient can be used as a parameter to compare specimens and has accurately and completely result than the other parameters.