The invention discloses a cotton endophytic bacterium HB3S-20 and application thereof to prevention and treatment of Verticilliumwilt of cotton, and belongs to the biological prevention and control of plant diseases. The endophytic bacterium is collected in CCTCC (China Center for Type Culture Collection) on November 1st, 2016, and is identified to be Pseudomonas-putida-HB3S-20; the preservation number is CCTCC NO: M2016608. The strain HB3S-20 can be used for the biological prevention and treatment of Verticilliumwilt of cotton. Under the greenhouse condition, the strain HB3S-20 has the average prevention effect of 62.5 percent on the Verticilliumwilt of cotton; good biological prevention activity is shown; no adverse influence exists on the cotton emergence rate and the cotton growth; for the cotton treated by the HB3S-20 bacterium solution, the plant height of the cotton plant and the fresh weight are obviously increased through being compared with blank control.