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Abstract Food quality monitoring, particularly, the detection of bacterial pathogens and spoilage throughout the food supply chain, is critical to ensure global food safety and minimize food loss. Incorporating sensors into packaging is promising, but it is challenging to achieve the required sampling volume while using food‐safe sensor materials. Here, by leveraging water‐based processing of silk fibroin, a platform for the detection of pathogenic bacteria in food is realized using a porous silk microneedle array; the microneedle array samples fluid from the interior of the food by capillary action, presenting the fluid to polydiacetylene‐based bioinks printed on the backside of the array. Through the colorimetric response of bioink patterns,Escherichia colicontamination in fish fillets is identified within 16 h of needle injection. This response is distinct from spoilage measured via the increase in sample pH. It is also shown that the microneedles can pierce commercial food packaging, and subsequently sample fluid and present it to the sensor, enabling the adaptation of the technology downstream in food supply chains such as in stores or at home. This study highlights that regenerated structural biopolymers can serve as safe materials for food contact and sensing with robust mechanical properties and tailored chemistry.