DNA methylation is an important epigenetic phenomena and plays crucial roles in the gene regulation. Many studies showed that DNA methylation can be used as clinical diagnostic biomarker. However, the ability to detect the DNA methylation status quickly and accurately is a prerequisite and key point for clinical application. By using two kinds of primers which can bind to methylated and unmethylated template respectively, methylation specific PCR (MSP) can distinguish DNA methylation status and prove to be a feasible and convenient diagnostic technique in clinical practice. Unlike traditional PCR, MSP mainly has four difficulties: how to enhance the specificity of binding to primer-methylated/unmethylated template, how to reduce the difference of Tm value of primer sequences, how to remove false positive amplification and how to improve sensitivity. Though most MSP primer design tools have proposed various solutions for those difficulties, there are still some defects in consideration of primer influencing factors, multitasking, prediction of specific amplification in MSP primer design and evaluation. Therefore, in this study, after deep exploration of existing MSP primer design tools, a novel MSP primer design and graphic evaluation tool named MethyScan was developed based on the integration of Bowtie, SAMtools, and BEDTools with Python graphic library Matplotlib and third-party functional libraries Biopython and Primer3-py. Three functional modules were involved in MethyScan including primer design, genome indexing and primer evaluation. MethyScan not only has the ability to perform MSP primers design and Nested primers adaptation, but also can evaluate primers specific/non-specific amplification with the analysis of primer binding information on four converted genomic templates and graphically displaying of the difference between non-specific amplification and target. Meanwhile, the comparison of MSP primer design for six potential biomarkers TFPI-2, NDRG4, CDKN2A, CD44, CASP8, and SDHD in esophageal cancer, colorectal cancer, and other malignant tumors suggested that MethyScan can not only obtain primers with more CpG sites, but also obtain primers with same or similar locations to those of other softwares, and the difference of Tm values of primers is even smaller. As the first MSP primer design tool for graphically displaying specific/non-specific amplification differences, MethyScan can effectively improve the accuracy of methylation primer design and provides strong support for the development of clinical DNA methylation detection projects, tests and diagnostic kits. The download address of MethyScan is: https://github.com/bioinfo-ibms-pumc/MethyScan.