ALKBH3 is an important marker for early diagnosis and histopathological grading of prostate cancer. However, the lack of a rapid and sensitive method to quantify the enzyme's activity in the current time necessitates the development of a new quantitative assay. Herein, we first tried to quantitative assay for ALKBH3 activity using an electrochemical method based on the degradation of the signal probe due to alkyl group of the m1A removal by ALKBH3. A strong electrochemical signal can be obtained when the ferrocene (Fc) labeled dsDNAs with 1-methyladenine are immobilized on the electrode. In the presence of ALKBH3, the 3′ blunt of DNA can be formed because of the removal of alkyl group of the Fc-DNA probe, which can be recognized and degraded by Exonuclease III (Exo III). As a result, the electrochemical signal produced by Fc greatly decreases, and the activity of ALKBH3 can be easily detected via changes in electrochemical signal. Quantitative analysis of ALKBH3 activity showed a wide detection range (0.1 and 20 ng/mL) and low detection limit (0.04 ng/mL). Furthermore, the method can be applied to detect 1-methyladenine through ALKBH3 in cell lysates and tissue samples, providing a new method for clinical detection of prostate cancer.