Background: The Check-up 35 has been a key element of primary prevention in German general practice since 1989. However, evidence on its effectiveness in detecting target diseases remains limited. Therefore, we analyzed the detection rates of the target diseases of the Check-up 35 in a large cohort of persons treated in German primary care. Methods: In a retrospective cohort study using data from 1,216 German primary care practices (IQVIA™ Disease Analyzer, 2019–2023), we compared Check-up 35 participants with non-participants via 1:5 propensity score matching. We analyzed diagnoses of arterial hypertension, diabetes mellitus, dyslipidemia, and chronic kidney disease (CKD) within three months post-check-up versus a random visit in controls. Medication prescriptions following diagnosis served as a secondary outcome. Results: We compared 51,829 participants with 259,145 matched non-participants. Participation was associated with higher diagnosis rates: hypertension (HR 1.56), diabetes (HR 1.69), dyslipidemia (HR 3.24), and CKD (HR 2.07). The effect was especially pronounced for dyslipidemia in individuals under 40 (HR 5.10). Prescription rates after diagnosis were similar or lower in the Check-up group. Conclusion: Check-up 35 is effective in early detection of key chronic diseases. Broader participation should be encouraged to improve prevention.