Rapid and accurate detection of Streptococcus pneumoniae (Sp), Haemophilus influenzae type b (Hib) and Mycobacterium tuberculosis complex (MTBC) in sputum by conventional methods remains problematic. Primers based on capsular polysaccharide biosynthesis gene (cpsA), the region II of the capsulation locus (cap), the insertion sequence IS6110 were designed for Sp, Hib, MTBC respectively. These primers were incorporated in a multiplex touchdown PCR assay for simultaneous detection of Sp, Hib and MTBC. The multiplex touchdown PCR assay was evaluated using standard strains and clinical sputum samples. The multiplex touchdown PCR assay showed 100% specificity in identifying Sp, Hib, MTBC from pure culture of standard strains. The sensitivities of the multiplex touchdown PCR assay were 94%, 98%, 98% for detection of Sp, Hib and MTBC respectively based on culture results while evaluated using 492 consecutive qualified clinical sputum samples; the specificities were all 100%. This highly sensitive and specific multiplex touchdown PCR assay offers a rapid and simple method for detection of Sp, Hib and MTBC in clinical sputum samples.