Bacillus subtilis as an important host has been widely used in synthetic biology, metabolic engineering, and production of industrial enzymes. To fully take advantage of this organism, 114 endogenous putative promoters were measured with a green fluorescent protein reporter and four classes of phase-dependent promoters (class I: exponential phase; class II: middle-log and early stationary phases; class III: lag-log and stationary phases; class IV: stationary phase) with different strengths were identified. The transcriptional strengths ranged from 0.03 to 2.03-fold of that of the commonly used strong promoter P-43. On this basis, the temperature (for instance P (bltD) , P (ydaD) , and P (gerBC) ) and pH (such as P (abrB) , P (ydjO) , and P (opuE) ) inducible phase-dependent promoters were further identified and characterized. In comparison, P (abrB) (class I), P (spoVG) (class II), and P (lytR) (class III) achieved the highest expression levels of esterase, keratinase, and alkaline polygalacturonate lyase, respectively. The constructed phase-dependent promoter library should have great application potentials for enzyme production, metabolic engineering, and synthetic biology.