This study examines the relationship between mental health and employment development among pre-service biology teachers, conducting a three-month longitudinal survey of 259 undergraduates across grade levels, yielding 771 valid questionnaire responses. Assessments encompassed emotional profiles, stressor types, interpersonal dynamics, emotion regulation strategies, sleep quality, and support requirements. Results revealed a complex yet predominantly stable and positive overall psychological state among participants. Primary stressors included future uncertainty (intensifying with academic progression) and academic pressure (diminishing across grades). Over half of the students reported moderate sleep quality, characterized by occasional insomnia or frequent dreaming, with sleep quality showing a significant correlation with emotional states. Emotion regulation strategies were diverse, featuring coexisting adaptive and maladaptive approaches with distinct grade-based variations. Interpersonal relationships were generally satisfactory, though support needs varied by grade, peaking in the junior year and declining in the senior year. Key contradictions emerged: high self-evaluation coexisted with stress and low help-seeking tendencies; maladaptive emotion regulation correlated with poor sleep quality; and senior students’ future planning stress was linked to both sleep disturbances and emotional distress. In light of these findings, the study proposes implementing a multidimensional mental health intervention framework, enhancing the integration of employment guidance and academic education, and refining career development systems to comprehensively safeguard students’ mental well-being and facilitate their employment progression.