The study aims to increase the accuracy and stability of the oxidase test due to the frequent false-positive and false-negative to the oxidase test during the microbiology experimental teaching. Here, we optimized experimental procedure and tested various culture mediums for pseudomonas aeruginosa by evaluating various bacterial culture medium and oxidase test kit to find a proper oxidase test protocol. Colonies were picked up with a sterilized toothpick, glass stick, or directly picked up with the paper disk. As a result, pseudomonas aeruginosa cultured in a slant agar medium frequently showed false positivity in the oxidase test, whereas pseudomonas aeruginosa cultured in regular agar plate or blood agar plate didn’t show any false positive. The experiment results showed that the blood agar was the best medium for bacterial culture for the oxidase test, and the oxidase test paper disk or N,N-Dimethyl-p-phenylenediamine dihydrochloride test paper showed a similar result in the oxidase test.