Standardized and well-characterized genetic building blocks allow the convenient assembly of novel genetic modules and devices, ensuring reusability of parts and reproducibility of experiments. In the first Bacillus subtilis-specific toolbox using the BioBrick standard, we presented integrative vectors, promoters, reporter genes and epitope tags for this Gram-positive model bacterium. With the Bacillus BioBrick Box 2.0, we significantly expand the range of our toolbox by providing new integrative vectors, introducing novel tools for fine-tuning protein expression, and carefully evaluating codon-adapted fluorescence proteins in B. subtilis, which cover the whole spectrum of visible light. Moreover, we developed new reporter systems to allow evaluating the strength of promoters and ribosome binding sites. This well-evaluated extension of our BioBrick-based toolbox increases the accessibility of B. subtilis and will therefore promote the use of this model bacterium and biotechnological workhorse as a host for fundamental and applied Synthetic Biology projects.