Overcoming the intrinsic low activity of most peroxidase mimics under neutral pH is crucial but still extremely challenging for the detection of disease markers in biological samples. Here, we chose nanoclay (i.e., montmorillonite K10, MK10) as a carrier to modulate the structure of Fe 1−x S nanozyme components through an interfacial modulation strategy, aiming at breaking the neutral pH limitation of Fe 1−x S. MK10 with abundant hydroxyl groups on its surface acts as a carrier to increase the ratio of Fe(II) and S(II−) content in surface Fe 1−x S. We verify that Fe(II)-promoted surface hydroxyl radical generation and S(II−)-promoted regeneration of Fe(II) play key roles in endowing peroxidase-like activity to Fe 1−x S at neutral pH. As expected, Fe 1−x S/MK10 exhibited 11-fold higher V max and 52-fold higher catalytic efficiency than bare Fe 1−x S. As a proof of concept, the sensor constructed based on Fe 1−x S/MK10 achieved colorimetric detection of xanthine under neutral conditions with a linear range of 5−300 μM and a limit of detection of 2.49 μM. Finally, we achieved highly sensitive detection of xanthine in serum using the constructed biosensor. Our contribution is the novel use of a nanoclay-mediated interfacial modulation strategy for boosting the peroxidase-mimicking activity and breaking the pH limitation, which contributes to the in situ detection of disease markers by nanozymes under physiological conditions.