The invention discloses a method of screening a 1-deoxyxylulose-5-phosphate racemase inhibitor from a marine microorganism fermentation broth. The method includes: 1) a step of preparing a marine microorganism fermentation broth sample, namely, a step of inoculating a fungus, fermenting, extracting metabolites, concentrating and eluting; 2) a step of designing a primer and performing PCR amplification by adopting the whole genome of vibrio vulnificus as a template, cloning a vector, converting escherichia coli, inducing expression of a target protein, and subjecting the target protein to elution, dialysis, ultrafiltration and desalting to obtain His-DXR; 3) a step of reacting a bacterial suspension, a resazurin solution and the marine microorganism fermentation broth sample obtained by the step 1), measuring the fluorescence value, calculating the inhibition rate through an equation, and screening the fraction the inhibition rate of which is higher than 80%; and 4) reacting the fraction screened in the step 3) and the His-DXR obtained in the step 2), calculating the inhibition rate, and screening the fraction the inhibition rate of which is not less than 50%. The method is simple and accurate, and lays a foundation for development of novel antibacterial medicines adopting Dxr as a therapeutic target or development of leading compounds.