In this study, an efficient mutagenesis and rapid screening method of high-yield gellan gum mutant by atmospheric and room temperature plasma (ARTP) treatment combined with Near-Infrared Spectroscopy (NIRS) was proposed. A NIRS model for the on-line detection of gellan gum yield was constructed by joint interval partial least squares (siPLS) regression on the basis of chemical determination and NIRS acquisition of gellan gum yield. Five genetically stable mutant strains were screened using the on-line NIRS detection of gellan gum yield in the fermentation from approximately 600 mutant strains induced by ARTP. Remarkably, compared with the original strain, the gellan gum yield of mutant strain 519 was 9.427 g/L (increased by 133.5%) under the optimal fermentation conditions, which was determined by single-factor and response surface optimization. Therefore, the method of ARTP mutation combined with the NIRS model can be used to screen high-yield mutant strains of gellan gum and other high-yield polysaccharide strains.