In this work, a novel type of hollow mesoporous silica nanoparticle (HMSN) with a rough surface has been successfully prepared via a facile soft-hard template route by using a carbon nanosphere as a hard template and cetyltrimethylammonium bromide (CTAB) as a soft template, respectively. This method involves the preparation of a carbon nanosphere, sequential coating of double SiO layers, and the removal of the inner carbon core and CTAB to produce HMSNs. The obtained HMSNs possess spherical morphology, a mesoporous shell, and crumpled surfaces. The controlled experiments prove that the addition of 3-ammonia propyl triethoxy silane (APTES) is very crucial for the formation of desired HMSNs. The cell tests indicate that HMSNs show a good biocompatibility. As a result, the potential applications of HMSNs are further explored for drug delivery and protein adsorption, using doxorubicin hydrochloride (DOX) and Cytochrome c (Cyt c) as the model drug and protein, respectively. The HMSNs exhibit high drug loading and protein adsorption capacity, as well as the controlled pH-responsive release behavior for DOX. Therefore, the HMSNs prepared are ideal candidates for various applications such as nanoreactors, drug delivery and protein adsorption.