There is increasing interest in the use of nanoparticles (NPs) for biomedical applications. In particular, nanobiophotonic approaches using fluorescence offers the potential of high sensitivity and selectivity in applications such as cell imaging and intracellular sensing. In this review, we focus primarily on the use of fluorescent silica NPs for these applications and, in so doing, aim to enhance and complement the key recent review articles on these topics. We summarize the main synthetic approaches, namely the Stöber and microemulsion processes, and, in this context, we deal with issues in relation to both covalent and physical incorporation of different types of dyes in the particles. The important issue of NP functionalization for conjugation to biomolecules is discussed and strategies published in the recent literature are highlighted and evaluated. We cite recent examples of the use of fluorescent silica NPs for cell imaging in the areas of cancer, stem cell and infectious disease research, and we review the current literature on the use of silica NPs for intracellular sensing of oxygen, pH and ionic species. We include a short final section which seeks to identify the main challenges and obstacles in relation to the potential widespread use of these particles for in vivo diagnostics and therapeutics.