New digital tools offer biodesign with unprecedented opportunities for monitoring, fabricating, iterating, and scaling designs. Serving aligned purposes and beyond, the Digital Twin (DT) is an emerging concept in bio-industries including bioprocessing and agri-food. In these fields, DTs enable comprehensive digital representations of a living system or process with continuous bidirectional connection to the physical world. Despite the concept’s potential to address certain challenges of uncertainty in designing (with) living systems, its applications and implications in biodesign and research remains largely underexplored. To bridge this gap, we propose a conceptual framework that synthesizes existing instantiations of DTs within bio-industries through a data flow lens. We mobilize the framework through a proof-of-concept DT for biofabrication with mycelium, by unpacking the data flows within the identified design and operation cycles. We conclude with a discussion on how DT capabilities respond to uncertainties in designing with living systems, limitations and future work.