The core challenge of in-service welding repair of oil and gas pipelines is the risk control of burn-through. Current research primarily focuses on macroscopic phenomena, lacking a systematic multi-scale analysis of burn-through mechanisms and their dynamic evolution. Existing criteria are primarily based on qualitative experience, and widely accepted quantitative safety assessment standards have yet to be established. Furthermore, insufficient understanding of multi-scale damage failure mechanisms and weak theoretical foundations have become bottlenecks in this field. This study targets X65 pipeline steel and combines in-service welding experiments with in-situ scanning electron microscope tensile tests to elucidate the formation mechanism of burn-through from a multi- scale perspective. The results show that during in-service welding, the remaining wall thickness of the pipeline continuously decreases with the welding process, ultimately resulting in burn-through holes. On one hand, the welding arc drives the expansion of the hole; on the other hand, the internal pressure of the medium further enlarges the hole, leading to the expulsion of water and rapid pressure loss in the pipeline. Notably, the fusion zone behind the maximum melt depth is subject to high temperatures, which reduces strength and degrades plasticity, exhibiting significant plastic strain, making it a high-risk area for burn-through instability. Before instability occurs, this region shows evident grain coalescence, with plastic deformation primarily occurring through dislocation slip; when the difficulty of activating slip systems increases, twinning deformation may be induced, and large twin grains rarely develop cracks. Strain concentration and crack initiation are more likely to occur between grains with significant orientation differences.