Silica chemistry provides pathways to uniquely tunable nanoparticle platforms for biological imaging. It has been a long-standing problem to synthesize fluorescent silica nanoparticles (SNPs) in batch reactions with high and low fluorescence intensity levels for reliable use as an intensity barcode, which would greatly increase the number of molecular species that could be tagged intracellularly and simultaneously observed in conventional fluorescence microscopy. Here, employing an amino-acid catalyzed growth, highly fluorescent SNP probes were synthesized with sizes <40 nm and well-separated intensity distributions, as mapped by single-particle imaging techniques. A seeded growth approach was used to minimize the rate of secondary particle formation. Organic fluorescent dye affinity for the SNP during shell growth was tuned using specifics of the organosilane linker chemistry. This work highlights design considerations in the development of fluorescent probes with well-separated intensity distributions synthesized in batch reactions for single-particle imaging and sensing applications, where heterogeneities across the nanoparticle ensemble are critical factors in probe performance.