The invention relates to a microbial synthesis preparation method and an application of cotton red nano pigment dyeing liquor. The microbial synthesis preparation method comprises the following steps: (1) producing the red nano pigment dyeing liquor by conducting fermentation through high-yield serratia marcescens: inoculating the serratia marcescens to an improved fermentation medium for 0-48h, adding a nonionic surfactant system, and conducting cultivation in a dark place at 25-28 DEG C for 50-100h at a shaking table rotation speed of 180-250rpm; and after conducting the fermentation, conducting centrifuging so as to remove the bacterium body (the serratia marcescens), so that the red nano pigment dyeing liquor is obtained; and the application comprises the following steps: (2) when the dyeing liquor is used for dyeing a cotton fabric, adding a dyeing assisting system to the dyeing liquor, regulating pH value to 6.0-8.0 and conducting dyeing at 60-100 DEG C for 30-90min by a bath ratio of 1 to (10-60), and finally conducting soaping and rinsing. With the application of the red nano pigment dyeing liquor provided by the invention, the problem that a water-insoluble microbial pigment cannot be used for dyeing the cotton fabric is solved. The dyed cotton fabric is excellent in color fastness, bright in color and excellent in antibacterial performance; and inhibitory rates of the nano pigment dyeing liquor on escherichia coli and staphylococcus aureus reach 70% and 95% or above; therefore, the the nano pigment dyeing liquor has a broad application prospect.