Silk fibroin (SF) has demonstrated great potential in diagnostics due to its excellent biocompatibility and biodegradability. Used as microneedles (MNs), SF is a compelling option for constructing an adaptable catalyst diagnostic platform due to its high integration. However, the defective hardness and swelling of SF MNs confine further catalyst-based visional detection. Herein, we present a facile approach involving the β-sheet induction by a nonionic surfactant, Tween 20, posing an on-demand regulation of SF MNs for further visual detection. Compared with the usual nonionic surfactants (Tween 80, PEG, and PEGMSE), Tween 20 demonstrated a remarkable ability to induce β-sheet formation within SF, which subsequently could facilitate the distinctive hardness (>1.06 N/needle) and swelling (60−180%) for SF MNs. Then, the agarose-based glucose model was used for visual detection, and consequently, the glucose oxidase (GOx)-HRP-TMB-integrated SF MN patch demonstrated a significant colorimetric quantitation as glucose concentration within 25−600 mg/dL (R 2 = 0.99304). More generally, the visual detection of glucose in types of fruits revealed the high efficacy and accuracy of the SF MN patch (R 2 = 0.94724), further indicating the advantage of Tween 20-tailored SF MNs as a visual platform. These achievements provide a facile tactic to reach an alternative hardness and swelling regulation by one step for catalyst implanted based SF MNs, further revealing the great potential of SF MNs for visual diagnostic.