Environmental and process control applications have needs for sensors that operate continuously or repeatedly, making them applicable to batch measurement and flowing product stream measurement. Additionally, for lactose monitoring in dairy-processing plants, the sensors must have sufficient flexibility to handle a wide range of substrate concentration and be resilient to withstand wide pH excursions brought about by frequent exposure to clean-in-place chemicals that happen without any warning. This paper describes the development and trialling of an at-line lactose biosensor that meets the needs of the dairy industry for loss monitoring of lactose in dairy-processing plants by the combination of a third-generation enzyme biosensor with a sequential injection analyser. Results, both from grab sample analysis and an at-line factory prototype, are shown from their operation when installed at a Fonterra dairy factory (New Zealand) during the 2011–2012 season. Previous sensor fabrication methods were converted to a single-step process, and the flow-through cell was adapted to bubble-free operation. The lactose concentration in wastewater-processing streams was successfully monitored by taking and analysing samples every 2–3 min, semi-continuously, for 3 months by an unskilled operator. The Fonterra site flushes approximately 100–300,000 L of wastewater per hour from its lactose plant. In the 2011–2012 season, the daily mean lactose content of this wastewater varied significantly, from 0.0 to 8.0 % w / v (0–233,712 μM) and equated to substantial total losses of lactose over a 6-month period. These lactose losses represent lost saleable or useable product.