Setting a reference scale for your 2D map fixes the size of symbols and text to the desired height and width at that scale. As you zoom in and out, your symbols and text will increase or decrease in size accordingly. When no reference scale has been set (the default behavior), symbol and text sizes remain the same on your map as you zoom in and out.
To set a reference scale, right-click the name of the map in the Contents pane and click Properties. On the Map Properties dialog box, on the General page, expand the Reference scale drop-down list and choose a value or type your own. To use the current map scale, open the map's context menu by right-clicking the name of the map and clicking Set Reference
Scale 
.
From this same menu, you can choose Clear Reference
Scale 
or Zoom To Reference Scale 
to return the view from other map scales.
Setting a reference scale is useful if you want the detail in your map to look the same as on-screen when printed. Using a reference scale means the map symbols appear on-screen at the size they will appear in the printed copy. For example, if you are creating a map for publication that will be printed at a scale of 1:25,000, set the reference scale to 1:25,000. You are now able to zoom in on areas with a lot of detail and labeling and assess whether you want to adjust symbol sizes or text.
When a reference scale is set, all feature symbology and labels in the current map are scaled relative to the reference scale by default. However, you can disable scaling for individual layers by double-clicking the name of the layer in the Contents pane to open the Layer Properties dialog box, clicking the Display tab, and unchecking Scale symbols when a reference scale is set.
Note:
Because annotation and dimension features have their own reference scales, they are not affected by the map's reference scale.
While reference scales are useful for designing printed maps, it's likely that you will want to disable this reference scale behavior when working with interactive and web maps. Typically, a multiscale web map will use symbol and text sizes scaled uniquely to each map scale.