This work presents the morphological and optical properties of luminescent silica spheres, discussing applications in bioimaging and biosensing. The spheres are obtained by the hydrolysis and condensation of tetraethylorthosilicate (TEOS) and can be synthesized by following either a basic or an acidic route. Luminescence emission is induced after incorporation of aminopropyltriethoxysilane (APTES) during synthesis or by introducing an optically active element, such as erbium, or other rare-earth elements. The luminescence properties of APTES-functionalized silica spheres have been investigated and optimized by varying the annealing temperature. On the other hand, erbium incorporation in silica spheres was also studied and the corresponding Er3+ luminescence emission at 1.54 μm was evaluated for intensity and lifetime. The basic pH environment in the synthesis allows good control of the size of the spheres (∼200 nm in diameter), whereas the acidic route produces a wide dispersion in particle size (200–5000 nm). Both these approaches, however, can be followed to obtain an efficient photoluminescence (PL) emission for the APTES-functionalized silica spheres after 400–600°C thermal treatment. If Er(NO3)3 is introduced in the basic solution, a rapid precipitation of Er(OH)3 occurs, but erbium can be easily and efficiently incorporated in the acid-synthesized spheres, showing high PL intensity at 1.54 μm with lifetime of 3.9 ms. Finally, I discuss perspectives for the applications of these luminescent silica spheres, in particular as biological markers for bioimaging and biosensing.