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In microbiological research, traditional methods for bacterial screening and antibiotic susceptibility testing are resource‐intensive. Microfluidics offers an efficient alternative with rapid results and minimal sample consumption, but the demand for cost‐effective, user‐friendly platforms persists in communities and hospitals. Inspired by the Magdeburg hemispheres, the strategy adapts to local conditions, leveraging omnipresent atmospheric pressure for self‐sealing of Rotation‐SlipChip (RSC) equipped with a 3D circular Christmas tree‐like microfluidic concentration gradient generator. This innovative approach provides an accessible and adaptable platform for microbiological research and testing in diverse settings. The RSC can avoid leakage concerns during multiple concentration gradient generation, chip‐rotating, and final long‐term incubation reaction (≥24 h). Furtherly, RSC subtypes adapted to different reactions can be fabricated in less than 15 min with cost less than $1, the result can be read through designated observational windows by naked‐eye. Moreover, the RSC demonstrates its capability for evaluating bacterial biomarker activity, enabling the rapid assessment of β‐galactosidase concentration and enzyme activity within 30 min, and the limit of detection can be reduced by 10‐fold. It also rapidly determines the minimum antibiotic inhibitory concentration and antibiotic combined medications results within 4 h. Overall, these low‐cost and user‐friendly RSC make them invaluable tools in determinations at previously impractical environment. [ABSTRACT FROM AUTHOR]