With the ongoing digital transformation of global healthcare services, medical guidance apps have become essential tools for patients, particularly for facilitating online medical appointments. However, inadequate interface designs often lead to increased cognitive load and heightened user anxiety. This study examines the impact of interface interpretability and layout on users’ cognitive load and emotional response during online medical appointment. A 3 × 2 mixed-factorial experiment was conducted with 24 participants, employing Tobii eye-tracking technology, electrodermal activity (EDA) sensors, and the PAD emotion scale for subjective evaluation. The results indicate that interface interpretability significantly affects both cognitive load and emotional response. High-interpretability interfaces alleviate cognitive load, while medium-interpretability interfaces tend to increase it. The layout of the interface also influences task efficiency, with the B1 layout (collapsible dropdown style) proving more efficient in terms of visual positioning. Although the interaction between layout and interpretability was not significant in most aspects, it did influence emotional arousal. The combination of A3 interpretability design and the B1 layout was found to be optimal in terms of reducing cognitive load and enhancing emotional well-being. Based on these findings, strategies for optimizing interface design are proposed: (1) Precisely enhance interface interpretability. (2) Optimize high-interpretability interface layouts. (3) Match interpretability with layout style.