The present invention relates to biomedicine technology, and is especially process of screening phosphonomycin producing strain. The screening process includes: culturing strain to be identified with dextro phosphonomycin as substrate and colibacillus as the bioassay indicator; pre-cooling the biotransformed phosphonomycin fermenting liquid to 0-10 deg.c, setting the liquid in alcohol-ice cooling agent at -30 deg.c to -10 deg.c to freeze at vacuum degree of 10-20 Pa and temperature -30 deg.c to -10 deg.c for 15 min to 2 hr to form concentrate; and identifying whether to producing inhibition zone with Oxford cup on bioassay plate to screen biotransformed strain. The present invention has the advantages of no deactivation on antibiotic, simple operation, low cost, etc.