Detection of biomarkers associated with physiological conditions provides critical insights into healthcare and disease management. However, challenges in sampling and analysis complicate the detection and quantification of protein biomarkers within the epidermal layer of the skin and in viscous liquid biopsy samples. Here, we present the "Lab-on-the-Needles" concept, utilizing a microneedle patch-based sensing box (MNP-based SenBox) for mobile healthcare applications. This system facilitates the rapid capture of protein biomarkers directly from the in situ epidermal layer of skin or liquid biopsies, followed by on-needle analysis for immediate assessment. The integration of horseradish peroxidase-incorporated zeolitic imidazolate framework-8 (HRP@ZIF-8) as a sensitive and stable signal probe, the detection limit for anti-SARS-CoV-2 NP IgA antibodies and various SARS-CoV-2 S1P mutant strains improves by at least 1,000-fold compared to FDA-approved commercial saliva lateral flow immune rapid tests. Additionally, the MNP-based SenBox demonstrated minimally invasive monitoring and rapid quantification of inflammatory cytokine levels (TNF-α and IL-1β) in rats within 30 min using a portable ColorReader. This study highlights the potential of the MNP-based SenBox for the minimally invasive collection and analysis of protein biomarkers directly from in situ epidermal layers of skin or liquid biopsies that might facilitate mobile healthcare diagnostics and longitudinal monitoring.