To develop a host-vector system for heterologous protein co-expression and purification in the Trichoderma reesei (teleomorph Hypocrea jecorina) industrial strain RUT-C30. The co-expression and purification system is based on: (i) an efficient and reliable selectable marker using bar (phosphinothricin acetyltransferase gene); (ii) a compact hygromycin B resistance marker; and (iii) a versatile integration plasmid for gene expression and knockout. Fluorescent protein genes were introduced into T. reesei and the corresponding proteins were purified from fermentation broth. The host-vector system was used in a proof-of-principle approach to achieve the co-expression of an alkaline endoglucanase and an alkaline cellobiohydrolase. This protocol can be used at an industrial scale to produce large amounts of proteins in T. reesei.