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In this work, we reported a new strategy based on DNA strands hydrolysis in combination with the employment of functionalized carbon black modified screen‐printed electrode (f‐CB/SPE) for guanine detection. Using glass microbeads as a solid substrate of ssDNA probe fixation, and under appropriate conditions of temperature, time, and hydrochloric acid concentration, DNA hydrolysis has been successfully used to release the guanine, allowing its oxidation at f‐CB/SPE using differential pulse voltammetry. The selectivity was successfully tested with a non‐complementary sequence, and a detection limit of 0.7 nM of the target DNA has been reached. [ABSTRACT FROM AUTHOR]