Background Chronic neural recording in freely moving animals is important for understanding neural activities of cortical neurons associated with various behavioral contexts. In small animals such as mice, it has been difficult to implant recording electrodes into exact locations according to stereotactic coordinates, skull geometry, or the shape of blood vessels. The main reason for this difficulty is large individual differences in the exact location of the targeted brain area. New methods We propose a new electrode implantation procedure that is combined with transcranial flavoprotein fluorescence imaging. We demonstrate the effectiveness of this method in the auditory cortex (AC) of mice. Results Prior to electrode implantation, we executed transcranial flavoprotein fluorescence imaging in anesthetized mice and identified the exact location of AC subfields through the skull in each animal. Next, we surgically implanted a microdrive with a tungsten electrode into exactly the identified location. Finally, we recorded neural activity in freely moving conditions and evaluated the success rate of recording auditory responses. Comparison with existing method(s) These procedures dramatically improved the success rate of recording auditory responses from 21.1% without imaging to 100.0% with imaging. We also identified large individual differences in positional relationships between sound-driven response areas and the squamosal suture or blood vessels. Conclusions Combining chronic electrophysiology with transcranial flavoprotein fluorescence imaging before implantation enables the realization of reliable subfield-targeted neural recording from freely moving small animals.