Exhibit 23.1

CONSENT OF INDEPENDENT REGISTERED PUBLIC ACCOUNTING FIRM

We consent to the incorporation by reference in the following Registration Statements:

(1)Registration Statements (Form S-8 No. 333-256521, 333-263343, 333-268071 and 333-269727) pertaining to the 2021 Equity Incentive Plan, 2021 Employee Stock Purchase Plan and 2022 Equity Inducement Plan of Day One Biopharmaceuticals, Inc., and

(2)Registration Statement (Form S-3 No. 333-265346) of Day One Biopharmaceuticals, Inc.

of our report dated March 6, 2023, with respect to the consolidated financial statements of Day One Biopharmaceuticals, Inc., included in this Annual Report (Form 10-K) for the year ended December 31, 2022.

/S/ Ernst & Young LLP

San Mateo, California

March 6, 2023