The 21st century demands mastery of various skills, and one of them is a higher-order thinking skill. Specialized instruments are required to measure these skills to be aware of their level of achievement. This research is classified as development research to create tools capable of measuring higher-order thinking skills of pre-service biology teachers. The development procedure is consisting of four stages, including instrument design, instrument trial, determination of validity, and determination of reliability. The instrument developed consisted of 12 items, which were then validated by experts. Trial stage involving 110 pre-service biology teachers in the Biology Education Program of eleven March University. The tested instrument is analysed to determine its validity and reliability. The results of the construct validation show model classified as fit according to the indicator of each instrument. The Results of the items analysis showed all items have an excellent discriminating power index. Also, each piece also has a difficulty index that is classified as a medium. Based on the results, the instrument developed has the qualifications to measure the higher-order thinking skills (transfer of knowledge) of pre-service biology teachers.