Understanding the stick-slip instability of granular materials is crucial to studying the quasi-periodicity of fault slip. The macroscopic frictional responses have inferred that particle size is one of the notable variables. To reveal the micro-mechanical cause, we analyzed the frequencies of Acoustic Emission (AE) events induced by the glass bead deformation through the compression tests and examined the effects of particle diameter on the stick-slip mechanism. We found that AEs distribute in the three frequency bands during the direct shearing process, with the probability density reflecting the three-stage evolution of force chains. At the microscopic scale, particle friction within the force chains induces micro-failures, generating low-frequency (0–100 kHz) AEs, as evidenced by environmental scanning electron microscopy. Meanwhile, high-frequency (200–350 kHz) AEs concentrate at the yield points. The probability density of AE events was used to quantify force chain deformation. The results show a negative correlation between AE rate and amplitude, with the micro-mechanical cause attributed to fewer frictional failures and an increase in critical elastic stiffness. The established AE-based experimental framework can provide insights into the micro-mechanisms of stick-slip instability.