The use of nanotechnologies for therapeutic or diagnostic purposes to fight cancer increased considerably over the last few years. In this work, we prepare functionalised mesoporous silica nanoparticles encapsulating new biphotonic chromophores in order to develop original biological markers. The template-directed synthesis of mesoporous silica nanoparticles doped with water-soluble fluorescent dyes optimized for two-photon excitation is described. Two structurally related symmetrical two-photon dyes that possess pyridinium acceptor end-groups conjugated to a fluorenyl core are synthesized by Heck coupling. These dyes display bright fluorescence (Φ ≈ 0.35) under both one- (ε ≈ 6 x 10000 M-1 cm-1) and two-photon excitation (σ2 ≈ 1000 GM) and were successfully encapsulated in silica nanoparticles via immobilization through non-covalent interactions. The nanoparticles present a mean diameter of 100 nm and a hexagonal network of mesopores as shown by Transmission Electron Microscopy. Interestingly the photophysical characteristics of the dyes are retained upon their immobilization into the silica matrix, leading to fluorescent silica nanoparticles with giant/unprecedented TPA cross-section (10000000 GM). The two-photon dye-doped mesoporous silica nanoparticles have been conjugated with folic acid in order to obtain efficient nanotools for bioimaging of cancer cells. Aminopropyltriethoxysilane and a PEG (polyethyleneglycol) spacer are successively grafted on the nanoparticles. They are finally functionalised by folic acid. Since folic acid receptors are overexpressed on tumoral cells, the interactions between these new biological markers and tumoral cell cultures have been studied. Cytotoxicity studies performed with MCF7 and HeLa cancer cells demonstrate that these functionalized NPs are much less cytotoxic than the non-functionalized NPs against both cell lines. Such nanospheres represent attractive nanoplatforms for the development of biotargeted biocompatible luminescent tracers.