Sugar beet is the second biggest world contributor to sugar production and the only one grown in Europe. One of the main limitations for its competitiveness is the lack of effective tools for assessing sugar content in unprocessed sugar beet roots, especially in breeding programs. In this context, a dedicated near infrared (NIR) fiber-optic probe based approach is proposed. NIR technology is widely used for the estimation of sugar content in vegetable products, while optic fibers allow a wide choice of technical properties and configurations. The objective of this research was to study the best architecture through different technical choices for the estimation of sugar content in intact sugar beet roots. NIR spectral measurements were taken on unprocessed sugar beet samples using two types of geometries, single and multiple fiber-probes. Sugar content estimates were more accurate when using multiple fiber-probes (up to R2 = 0.93) due to a lesser disruption of light specular reflection. In turn, on this configuration, the best estimations were observed for the smallest distances between emitting and collecting fibers, reducing the proportion of multiply scattered light in the spectra. Error of prediction (RPD) values of 3.95, 3.27 and 3.09 were obtained for distances between emitting and collecting fibers of 0.6, 1.2 and 1.8 µm respectively. These high RPD values highlight the good predictions capacities of the multi-fiber probes. Finally, this study contributes to a better understanding of the effects of the technical properties of optical fiber-probes on the quality of spectral models. In addition, and beyond this specificity related to sugar beet, these findings could be extended to other turbid media for quantitative optical spectroscopy and eventually to validate considered fiber-optic probe design obtained in this experimental study.